W.H.O Guidelines.ppt by Dr.U.Srinivasa

Useful for B.Pharm students

2. W.H.O.GUIDELINES
Herbalmedicineisbeingusedbyabout80%
oftheWorldpopulationforprimaryhealthcare.
Theyhavefaith,culturalacceptabilityand
beingnaturalbelievedtohavebetter
compatibilitywiththehumanbody.

3. •MajorGoalsandObjectivesoftheWHO
Guidelinesare
•Topromotetheappropriate,safeandeffective
useofherbalmedicinesandtoencouragethe
integrationofherbalmedicinesintothe
mainstreamofmedicalsystems

4. •AWHOguidelineinvolvespolicesonQuality,
SafetyandEfficacyassessmentofcrudeplant
materialsandstabilityoffinishedproducts.
•Itisalsogivingalotofemphasison
documentationoftraditionaluseofitsactivity
determination(animal,human)andsafetybased
onexperienceor/toxicologystudies

5. DEFINATION
Drugevaluationisdefinedasthedetermination
ofidentity,purityandqualityofadrug.
Identitymeanstheidentificationofdrugs.
Qualitymeansthequantityofactive
constituents.
Puritymeanstheextentofforeignmaterial
present.

6. NEED OF STANDERDIZATION
1. Biochemical and Geographical variation in the drug
2. Deterioration during treatment and storage.
3. Substitution and adulteration
4. Export of quality of natural drugs
5. Popularity of natural drugs for primary care in
developing countries

7. 6. Herbal medicines ,however are not necessary always safe
simply because they are natural. Some have given rise to
serious adverse reactions and some contain chemicals that
may produce long term side effects such as Carcinogenicity
and Hepatotoxicity.
7.Alsoherbalmedicinewillonlybenefitthehealthofhuman
beingswhentheyofgoodquality
8.Furthermorewithincreaseduseofbothherbalmedicine
andmodernwesternpharmaceuticaldrugs,thereisa
needtomonitorinteractions

8. SIGNIFICANCES OF DRUG EVALUATION
1.Itishelpfulforthedetectionofadulterants,ifitis
adulterated.
2.Itisalsohelpfultodetectthepresenceofchemical
constituentspresentinthecrudedrug.

9. WHO PARAMETERS FOR STANDARDIZATION OF HERBAL
RAW MATERIAL, EXTRACTS AND THEIR PRODUCTS
(METHODS OF DRUG EVALUATION )
1. Preliminary evaluation
2. Morphological evaluation
3. Microscopical evaluation.
4. Physical Qualitative evaluation
5. Physical Quantitative evaluation
6. Chemical evaluation
7.WHO Biological evaluation
8. Toxicogical evaluation
9. Pharmacological evaluation
10. Analytical evaluation

10. Physical Qualitative evaluation
1. Melting point
2.Solubility
3.Optical rotation
4.Viscosity
5. Refractive index
6. Boiling point
7. Chromatographic
8.Spectroscopic evaluation

11. MELTING POINT
•Incaseofpurechemicalsorphytochemicals
meltingpointsareverysharpandconstant.
•Sincethecrudedrugsfromanimalorplantorigin
containthemixedchemicals,sotheyare
describedwithcertainrangeofmeltingpoints.
•Colophony–75-85,Beeswax-62-65
•Woolfat-34-40

12. SOLUBILITY
•Thepresenceofadulterantinadrugcouldbe
indicatedbysolubilitystudies
•Eg;Castoroilissolublein3volumesof90%
alcohol,whileadulteratedformmayshowgood
solubilityinalcohol

13. OPTICAL ROTATION
•Certaindrugsarefoundtohavethepropertyofrotating
theplaneofpolarizedlightinthepurestateorin
solution. Thus,theyaredescribedtobeoptically
activeandthispropertyisknownasopticalrotation.
•Dextro-rotatory-towardsright(+),Leavo-rotatory-
towardsleft(-)
•Opticalrotationisdeterminedat25˚Cdegreeusing
sodiumlampasthesourceoflight.
•Example-
•Carawayoil-+70˚to80˚
•Honey-+3˚to15˚

14. REFRACTIVE INDEX
•Whenarayoflightpassesfromonemediumto
anotherofdifferentdensity,itbentfromoriginal
path.
•Dependinguponpurity,itisconstantforaliquid
andcanbeconsideredasoneofthecriteriaforits
standardization.
•Examples–Arachisoil–1.4678-1.4698
•Carawayoil–1.4838-1.4858

15. CHROMATOGRAPHY
•THINLAYERCHROMATOGRAPHY
•Herecomparisonofretentionfactorvalues(Rf)
withstandardsascertainqualitativeevaluationof
constituents
Rfvalues–
Example:
Eugenol-0.47
Caravone-0.46
Borneol-0.24

16. THIN LAYER CHROMATOGRAPHY
•THINLAYERCHROMATOGRAPHY
•Herecomparisonofretentionfactorvalues(Rf)
withstandardsascertainqualitativeevaluationof
constituents
Rfvalues–
Example:Eugenol-0.47
Caravone-0.46
Borneol-0.24

18. SIGNIFICANCE:
1.Themethodisgenerallyusedtotestthepurityand
todetermineconcentrationofthedrug
2.Whenthequantityofthedrug/samplearevery
lessthenthedrugsareevaluatedbyspectroscopic
methods

19. SPECTROSCOPIC EVALUATION
•1.Colorimetricmethod
•2.Fluorimetricmethod
•3.Spectrophotometricmethod
•4.I.R.Spectrophotometricmethod
•5.NMRSpectroscopy
•6.MassSpectroscopy

20. PRINCIPLE:
•Thecapacityofcertainmoleculesofadrugtoabsorb
vibrationataspecificwavelengthisthebasisforthe
drugevaluation

21. •Procedure:
•Forthequantitativeevaluationofasubstancea
standardcurveisfirstpreparedbymeasuringthe
O.Dofaseriesofstandardsolutionsofthepure
compoundusinglightofasuitablewavelength,
usuallythatatwhichthecompoundgivesan
maximumabsorption.
•TheO.Dofthesolutiontobeevaluatedisthen
determined&itscompositionascertainedfromthe
standardcurve.

22. 1. COLORIMETRIC METHOD
•ItalsocalledasVisiblespectrophometry
•Visibleregionisfrom380-780nm
•Mostofthechemicalcompoundsgivecharacteristic
colorwithspecificreagent
•Theintensityofcolorinmanycasesaredirectly
proportionaltotheamountofconcentrationpresent

23. •Suchcompoundsareestimatedbycolorimetryata
suitablewavelength
•Eg:EphedrinetreatedwithNinhydrinereagentforms
violetcolor,theintensityismeasuredat550nm.
•Ergot(Totalalkaloids)whentreatedwithp-dimethyl
aminobenzaldehydereagent(PABA)formsviolet
color,theintensityismeasuredat550nm.

24. 2. FLUORIMETERIC METHOD
•TheinstrumentusedisFluorimeter
•Somecompoundsproducesfluorescencewhen
treatedwithspecificreagent
•Theintensityofthefluorescenceisdirectly
proportionaltotheconcentrationofdrugpresent
•The intensity of the fluorescence is measured by
Fluorimeter.
•Eg: Quinine when treated with 0.1 N Sulphuric acid
produces blue fluorescence the intensity is
measured at 450 nm

25. 3.U.V.SPECTROSCOPIC METHOD
•ItisalsocalledasU.V.Spectroscopy,U.V.region
from185-380nm
•Somecompoundshavingthecapacitytoabsorb
vibrationsatspecificwavelengths
•The intensityismeasured by using
Spectrophotometer
•Eg:Papaverinein0.1NHcl,theconcentrationofthe
drugismeasuredat310nm
•Eg:Strychininein0.1NHcl,theconcentrationofthe
drugismeasuredat254nm

26. 4. INFRARED SPECTROSCOPY (IR)
•ItisalsoknownasI.R.Spectroscopy
•Theregionfrom1430-910cm
-1
isfingerprintregion
•Theconstantlyvibratingmoleculesstretchand
bendtheirbondswithrespecttooneanother,by
absorbinginfraredlight
•Functionalgroupscanbeidentified

27. •OH-group absorbs at 3200 -3600 cm
-1
•C-H-group absorbs at 3100 -2800 cm
-1
•Carbonyl group at 1700 -1800 cm
-1
etc

28. 5.NUCLEAR MAGNETIC RESONENCE
•It is also called as NMR
•It is also called as H NMR and C NMR
•By this number of protons and carbons can be predicted
from spectrum

29. 6.MASS SPECTROSCOPY
•It also called as MS
•Here molecules are bombarded with electrons
•Molecules are ionized and broken up into fragments
(m/e)
•By this molecular weight, molecular formula can be
predicted from the spectrum

30. PHYSICAL QUANTITATIVE PARAMETERS
•Ash values
•Extractive values
•Moisture content
•Volatile oil determination

31. ASH VALUES
Theresidueremainingafterincinerationistheash
contentofthedrug.(inorganicsaltsofcarbonates,
phosphates,silicatesofsodium,potassium,
calciumandmagnesium)isknownasashcontent.
AshvalueisacriteriontojudgetheidentityOR
purityofthecrudedrug

32. TYPES OF ASH VALUES
1.Total ash value
2.Acid insoluble ash value
3.Sulphated ash value
4. Water soluble ash value

33. Total ash value:
Useful for detecting low grade products
Useful for detecting exhausted products
Useful for detecting excess of sandy
Useful for detecting earthy matter with drug

34. •Examples
•Aloes –Total ash content 5 %
•Clove -Total ash content 7 %
•Ginger -Total ash content 5 %
•Cardamom -Total ash content 6 %

35. DETERMINATION OF TOTAL ASH VALUE
1.Weighaccuratelyabout3gmsofthepowdereddrug
inataredsilicacrucible
2.Incineratethepowdereddrugbygraduallyincreasing
theheatuntilfreefromcarbonandcool.Keepitin
desiccators
3.Weightheashandcalculatethe%ofthetotalash
withreferencetotheairdriedsample

36. CHEMICAL EVALUATION
•Qualitative evaluation to detect different classes of
phytochemicals
•Quantitative determination of phytochemicals
•Assay

38. BIOLOGICAL EVALUATION
•Animal activity
•Animal organ
•Tissue activity

39. SIGNIFICANCE:
1.Themethodisgenerallyusedwhen
standardizationisnotdonesatisfactoryby
chemicalorphysicalmethods
2.Whenthequantityofthedrug/samplearevery
lessthenthedrugsareevaluatedbybiological
methods

40. Thesemethodsareperformedonlivinganimals,
isolatinglivingorganandtissue,animalpreparation,
andmicro-organism
(Bioassay)

41. Following method is used as
1.Anti inflammatory activity
2.Analgesic activity
3.Antipyretic activity
4.Anti ulcer activity
5.Antidiabetic activity
6.Anthelmintic activity on earth worms

42. 7.Cardiacactivity-onfrogandpigeon
8.Microbiologicalmethods-livingbacteria,yeast,molds
areusedfortheassayingvitaminsandtodetermine
theactivityofantibioticdrugs

44. MICROSCOPIC EVALUATION
•Qualitative histological evaluation of types and
arrangement of tissues
•Quantitative assessment of stomatal index and
stomatal number , palisade ratio , vein islet ,vein
termination and lycopodium method

45. MICROSCOPIC METHOD
•SIGNIFICANCES
•Usedfortheevaluationoforganizeddrugsbytheir
knownhistologicalcharacters
•Helpsforthestudyofconstituentsbyapplicationof
chemicalmethodstosmallquantitiesofdrugsin
powderedformorhistologicalsectionsofthedrug
(MicrochemistryorChemo-chemistry)

46. •Methods of microscopic evaluation
•1.Histology
•2.Chemomicroscopy
•3.Powder analysis
•4.Microscopical linear measurements
•5.Quantitative microscopy

47. 1.HISTOLOGY
Histologicalstudiesaremadefromverythinsectionsof
drugs.ThecharacteristicsofCellwalls,Cellconstituents,
Trichomes,Fibres,Vesselsetc.,canbestudiedindetails
Eg,LignifiedtrichomesinNuxvomica
Eg,Wartytrichomesinsenna

48. Eg,WavymedullaryraysinCascara
Eg,ThepowderedclovedonotcontainscleridesOR
Calciumoxalatecrystals,butbothofthemare
presentinpowderedclovestalk

49. •2.ChemomicroscopyORMicrochemistry
•Byusingsomesimpletestshelpsforthedetectionof
someconstituents
•Eg:Adropofphloroglucinolandconc.Hydrochloricacid
giveredstainwithLignin
•MucilageisstainedpinkwithRutheniumredsolution
•IodinesolutionstainsStarchandHemicelluloseblue
color

50. 3.Powderanalysis
Ithelpsinidentificationanddetectionofadulterant
Eg.RhubarbandGingerarecharacterizedbytheirnon
lignifiedvessels
Eg.VarietiesofAloesbythepresenceORabsenceof
CrystalsofAloin
Eg.PowderNuxvomicaisidentifiedfromlignified
trichomes

51. •4.MicroscopicLinearMeasurements
•Diameterofstarchgrainswillassistindistingushining
varieties
•Eg.Cassia–10micronsfromCinnamon-8microns
•Eg.Lengthandwidthofthephloemfibres
•Eg.Cinchona-580-1350micronslenght

52. •5.Quantitative microscopy
•It includes
•a) Stomatal Index and Stomatal number
•b) Vein islets number
•c) Palisade ratio

53. •StomatalIndex
•Itisthepercentagewhichthenumberofstomataformto
thetotalnumberofepidermalcellsiscalledstomatal
index.
•Stomatalnumber
•Itisaveragenumberofstomatapersquaremillimeterof
leaf

54. •B)Veinisletsnumber
•Itisdefinedasnumberofveinisletspresentin1square
millimeterofleafsurfacemidwaybetweenmidriband
margin
•C)Palisaderatio
•Itisaveragenumberofpalisadecellsbeneatheach
epidermalcell.Palisaderatiocanbedeterminedwiththe
powdereddrug.

56. CHEMICAL METHOD
•SIGNIFICANCE:
•1.Usedforthedeterminationofamountofsingleactive
constituentORthegroupofrelatedconstituentsinthe
samedrug
•2.Thepurityofcrudedrugsisascertainedbyquantitative
estimationofactiveconstituentspresentinthem

57. •Typesofchemicalevaluation
•1.Gravimetricmethod
•2.Volumetricmethod
•3.Acidvalue,Saponificationvalue,Iodinevalue,Ester
value
•4.Thinlayerchromatography
•5.Qualitativechemicaltests

58. •GRAVIMETRICMETHOD
•Principle:
Inthismethodknownamountofthedrugistaken,the
activechemicalconstituentsareisolated,driedtoa
constantweightandthenweightisdetermined.
Example:Totalalkaloidcontentinsolanaceousdrugs
Podophyllininpodophyllumroot
Balsamicacidinbalsamofperuandbalsamoftolu

59. •VOLUMETRICMETHOD
•Usedforthedeterminationofphysico-chemical
characteristicsofthefixedoilsandfats,includesAcid
value,Saponificationvalue,Iodinevalue
•Nonaqueoustitrationmethodsusedfortheanalysisof
organicacids
•Example;Eugenolincloveoil,Carvoneincarawayoiland
Dilloil,EphedrineinEphedrabynon–aqueoustitration

60. •THINLAYERCHROMATOGRAPHY
•Herecomparisonofretentionfactorvalues(Rf)
withstandardsascertainqualitativeevaluationof
constituents
Rfvalues–
Example:Eugenol-0.47
Caravone-0.46
Borneol-0.24

61. •QUALITATIVE CHEMICAL ANALYSIS
•By using some simple chemical tests for identifying
specific organic groupings which may be present in
any drug to which its therapeutic activity is
attributed.
•Alkaloids –Dragendroffs Test
•Glycosides/ Sugars –Molischs Test
•Steroids –Lieberman Burchard Test
•Anthraquinones –Borntrager Test

62. •Flavonoids –Shinoda Test
•Tannins –Ferric Chloride Test
•Reducing Sugar –Fehling Solution Test
•Deoxy sugars -Keller-kiliani Test
•Mucilage -Ruthenium red solution
•Proteins and Amino acids -Ninhydrin Test

64. MORPHOLOGICAL EVALUATION
•Qualitative evaluation of colour , odour, and
taste , shape, extra features

65. MORPHOLOGY
SIZE–ofplantpartslikerhizomes,roots,corms,
bulbs,tubers,fruit,seed,stemetc
SHAPE–oftheplantpartlikenapiform,
annulated,
COLOUR–therangeofcolourlikegreyishofnux
vomicaseeds
EXTERNALMARKINGS–likefurrows,wrinkles,
ridges,annular,outgrowthsetc.

66. ODOUR
Aromatic
Balsamic
Camphoraceous
Spicy
Pleasant
Irritating

67. TASTE
Bitter
Sour
Astringent
Pungent
Acid
alkaline

68. SOUND–to see the ripeness of the fruit /
seed etc
FRACTURE
Granular
Splintery
Smooth

69. PLANT PARTS
Bud, Flower, Fruit, Leaf, Root, Seed, Stem
HABIT & HABITAT
Annual, Aquatic, Biennial, Climber, Creeper,
Epiphyte, Habit (Herb, Shrub) Perennial, Tree
etc
PHYLLOTAXY
Alternate, Opposite, Whorled

70. APEX OF LEAF
Acuminate, Acute, Obtuse
MARGIN OF LEAF
Ciliate, Crenate, serrate, entire, undulate
SHAPE OF LEAF
Cordate, Lanceolate, Oblong, Ovate

71. TYPE OF LEAF
Simple, Compound, Pinnate, Palmate
VENATION OF LEAF
Parellel, Reticulate, Unicosate, multicosate
INFLORESCENCE
Cymose, RAcemose
FLOWERS
Epigynous, Hypogynous, Perigynous
FRUITS
Drupe, Berry

72. WHO BIOLOGICAL EVALUATION
•Swelling Index determination
•Foam Index determination
•Haemolytic Index determination

73. SWELLING INDEX DETERMINATION
Definition:
Theswellingindexisthevolumeinmltakenupby
theswellingof1gmofplantmaterialinwaterunder
specifiedconditions.
Significances:
Usefulintheevaluationofcrudedrugswithswelling
propertiesespeciallygumsandmucilage,pectinand
hemicelluloses
Usefulforthedetectionofpurityofthecrudedrug

74. DETERMINATION
1.Transfer1gmoftheseedstoa25mlstoppered
cylinder
2.Filluptothe20mlmarkonthecylinderwithwater.
Agitategentlyandoccasionallyduring24hours
andallowedtostand
3.Measurethevolumeoccupiedbytheswollen
seeds.
4.Swellingindex–Isapaghulaseedshouldbein
between10-14

75. FOAM INDEX DETERMINATION
•Thistestisusedtomeasurethefoamingabilityofan
aqueousdecoctionofsaponincontainingmedicinal
plantmaterialswhichpossessespropertytoform
persistentfoamwhenanaqueousdecoctionisshaken

76. DETERMINATION
•Prepare aqueous decoction of about 1gm of coarse
powder material in 100ml of water by boiling for 30
minutes.
•Cool and filter into 100ml volumetric flask and add
sufficient amount of water to make up the volume to
100 ml
•Now prepare 10 stoppered test tubes (height –16cm
and diameter –16mm) in series containing 1,2,3 up to
10ml and adjust the volume of the liquid in each test
tube with water to 10 ml

77. DETERMINATION
•Stopper the tubes and shake them in a lengthwise
motion for 15 seconds, 2 frequencies per second.
•Allow to stand for 15 minutes and measure the height
of the foam
•Foaming Index = 1000/ A
•A-Volume of decoction having exact 1cm height.
•Examples –Licorice, Diascorea

78. HAEMOLYTIC INDEX DETERMINATION
•Saponinshaveabilitytocausehaemolysiswhere
haemoglobindiffusesintothesurroundingmedium
throughachangeintheerythrocyticmembraneand
makesbloodaclearsolution

79. HAEMOLYTIC INDEX DETERMINATION
•Thehaemolyticactivityofplantmaterials,ora
preparationcontainingsaponins,isdeterminedby
comparisonwiththatofareferencematerial.
•Asuspensionoferythrocytesismixedwithequal
volumesofaserialdilutionoftheplantmaterialextract.
•Thelowestconcentrationtoaffectcomplete
haemolysisisdeterminedafterallowingthemixturesto
standforgivenperiodoftime.
•Asimilartestiscarriedoutsimultaneouslywith
saponin.