Most useful for pharmacy students

1. –
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2. QUININE
It is a quinoline alkaloid of cinchona bark.
The other important alkaloids of this drug
are quinidine, cinchonine, cinchonidine,
cinchonamine etc.,
B.S. It consists of dried inner bark of
C.Calisaya, C.succirubra, C.officinalis,
C.ledgeriana and hybrids of this. Family –
Rubiaceae

3. Quinine and quinidine are stereo-isomers .
Quinine is laevorotatory and quinidine is
dextrorotatory
Uses :
Quinine is antimalarial
Quinidine is a cardiac depressant therefore
used in cardiac arrythmias.

4. ISOLATION
1. The dry powder bark material is first well mixed
with about 30% of its weight of calcium hydroxide
or calcium oxide and sufficient quantity of sodium
hydroxide solution to make a paste. It is allowed to
stand for few hours.
2. The mass is then transferred to a Soxhlet
apparatus and extraction is carried out with
benzene.

5. 3.Subsequently the benzene extract is shaken
with successive portions of 5% sulphuric
acid.
4.The aqueous acid extract is adjusted the pH
6.5 with dilute sodium hydroxide, cool.
Crystals of neutral quinine sulphate are
formed.
5.These crystals are freed from cinchonine
and cinchonidine by repeated
recrystallization from hot water.

6. 6. Colouring matter is removed by activated
charcoal.
7.Quinine sulphate crystals are dissolved in
dil sulphuric acid and made alkaline with
ammonia. Initially amorphous quinine is
formed , which becomes crystalline .
8. Finally washed to remove sodium and
ammonium salts and dried to 45- 55 o C.

7. IDENTIFICATION
1. Chemical tests
2. Thin layer chromatography

8. T.L.C:
Adsorbent : Silica gel -G
Mobile phase – Chloroform – diethylamine
(9:1)
Detection – Spray with methanolic KOH ;
Sulphuric acid reagent or UV Light

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10. EPHEDRINE
It is an amino alkaloid ( Non –heterocyclic,
proto-alkaloid or a typical alkaloid )
present in various species of ephedra.
Chemically , it is 1-phenyl,1- hydroxy,-2-
methyl amino propane.

11. • B.S It consists of the dried aerial parts
of ephedra sinica, ephedra nebrodensis
and ephedra geridiana Family :
Ephedraceae
• The other important species are ephedra
intermedia, ephedra major, ephedra
alata.

12. USES :
Ephedrine is a bronchodilator and useful in
the treatment of asthma and allergic
conditions such as rhinitis, fever etc.
Used as mydriatic

13. ISOLATION :
1.Ephedra powder is first made alkaline with
sodium carbonate and then macerated with
benzene overnight.
2.Benzene layer is separated by filtration and
treated with Dilute HCL
3.The aqueous acid layer is separated and
made alkaline with solid potassium
carbonate.

14. 4. Then extracted with chloroform
5.The chloroform extract is evaporated to
dryness to get the residue. This is mixed
with oxalic acid and warmed.
6. Alkaloidal oxalates are formed (ephedrine
oxalate and pseudo ephedrine oxalate)

15. • Ephedrine oxalate is less soluble in cold
water , on cooling crystals of ephedrine
oxalate is formed.
• Pseudo-ephedrine oxalate is remains in
solution
• 7. ephedrine is liberated from its oxalate
by shaking with alkali solution and then
extracted with a mixture of chloroform and
ether (1:3) to get ephedrine.

16. • Identification :
• 1. A slightly alkaline solution of ephedrine
on warming with Ninhydrine solution forms
violet color.
• 2. Ephedrine gives pink to red coloration
with con.sulphuric acid and 3 to 4 drops of
40% formaldehyde solution.

17. • 3. Dissolve ephedrine in water and add dil
Hcl and test separately with 10% copper
sulphate solution and add 20% sodium
hydroxide solution. A purple or voilet
colour developed.
• This is the ether extractable .On shaking
with ether the organic layer is purple but
aqueous layer is blue

18. • ESTIMATION :
• 1 . Titration methods :
• A. Back titration method
• B. Non –aqueous titration method
• 2. Colorimetry

19. 1.Back titration method (I.P.1966)
Principle : Ephedrine can be estimated by
back titration method.
In the procedure , the accurately weighed
sample is treated with the known excess
quantity of acid and the excess acid is back
titrated with alkali.

20. • Procedure :
• Weigh accurately about 0.5 gms of sample
and dissolve in 5ml of ethanol (95%). Add
50ml of 0.1M Hcl and titrate with 0.1M
NaoH, using methyl red as indicator until a
yellow colour is produced.

21. • 2. Non –aqueous titration
• Procedure :
• Weigh accurately about 0.17 gms of
sample and dissolve in 10ml of Mercuric
acetate solution, by warming gently . Add
50 ml of Acetone and titrating with 0.1M
Perchloric acid , determining the end point
by Potentiometrically

22. • 3. By Colorimetry :
• PRINCIPLE :
• Being a secondary amine , ephedrine forms
a colored complex (400nm ) with Picryl
chloride in benzene

23. • Procedure :
• Take 1,2,3,4,5 and 6ml of 0.01% solution
of pure ephedrine in benzene into test
tubes and adjust the volume to 90ml with
benzene.
• Add 1ml of 0.3% solution of Picryl chloride
to each test tube and close tightly for 2
minutes for exactly.

24. • Place them in a water bath at 75-77 o C for 20
minutes.
• Remove the tubes from the water bath and
allowed to stand for 3 minutes and measure
the absorbance at 400nm against the blank
prepared by the same procedure by taking
90ml of benzene and 1ml of 0.3% solution of
Picryl chloride.

25. • Estimation of the sample :
• The isolated ephedrine sample is prepared in
benzene and treated with 1ml of 0.3%
solution of picryl chloride its absorbance is
measured by keeping in the water bath at
75-77 o C for 20 min. and then allowing to
stand for 3 min at room temperature.

26. • Calculation:
• The concentration of the ephedrine in
the sample is obtained by interpolation
of the standard curve

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28. ANDROGRAPHOLIDE
• It is the bitter principle of Kalmegh. It is
chemically a bicyclic diterpenoid lactone .
• Neo- andrograpolide and andrographisside
are the other important active
constituents of the plant

29. • Biological source : Kalmegh consists of
dried leaves of the plant known as
Andrographis paniculata
• Family : Acanthaceae
• Uses : Hepatoprotective , bitter tonic

30. ISOLATION
• PRINCIPLE :
• Isolation is based on solubility of
andrographolide, it is soluble in methanol
and ethanol

31. PROCEDURE
• 1.Extract the dried coarse powder
successively with petroleum ether (60-
80 o C) ,chloroform and methanol
• 2. Collect the methanol extract and
concentrate and treat his with charcoal
for 24hrs

32. • 3.Filter and collect the filtrate , keep this
filtrate for overnight for crystallization
• 4.Then reflux the recovered charcoal
with methanol , filter and add filtrate to
the mother liquor . Keep it for overnight
for crystallization.

33. • 5.Purify the combined crystals from
methanol to get andrographolide ( M.P
228-229 o C )

34. IDENTIFICATION
• Chemical test :
• Sample and few drops of solution of
copper acetate gives emerald green color
• By T.L.C :
• Adsorbent – Silica gel G
• Mobile phase –Chloroform: Methanol (7:1)

35. • Reference standard- Andrographolide 1mg
in 1.5ml of methanol
• Test sample – Extract in methanol
• Detecting reagent –
• 20% sulphuric acid in methanol, heat at
120 o C for 10 minutes

36. • Rf value of Andrographolide – 0.70,
brown color spot.
• ESTIMATION METHODS :
• 1.Gravimetric method
• 2.Colorimetric method
• 3.High Performance liquid
chromatography (HPLC)

37. • 1.Gravimetric method :
• Known weight of the drug is taken, the
active constituents are isolated, dried to a
constant weight , the weight is
determined .
• 2.Colorimetric method :
• Andrographolide gives red color with
alcoholic KOH, which is measured at 400-
800 nm

38. • 3.High Performance Liquid
Chromatography (HPLC)
• Column – 5µm Spherical silica gel (3mm X
15cm)
• Mobile phase – Chloroform – Methanol
(9:1)
• Flow rate – 0.7 ml/ minutes
• Detector – UV at 254nm
• Standard preparation – Known

39. • Sample preparation –
• Drug extracted with methanol, evaporated
and residue dissolved in methanol
( 50µg/ml )
• PROCEDURE : 10µg/ml of standard and test
preparations are subjected to HPLC , record
the chromatogram and calculate the
percentage (%) of andrographolide from the
respective peak areas.

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41. RUTIN
• There are around 200 types of
Quercetin, Flavanoid glycosides, among
this the rutin is the one of most
important type. It is chemically
Quercetin-3- rutinoside . On hydrolysis ,
it yields the aglycone quercetin and the
sugars glucose and rhamnose.

42. SOURCES OF RUTIN
• 1. Buck wheat ( Fagophyrum esculentum- Family
– Polygonaceae )
• 2. Rhubarb – ( Rheum emodi- Family -
Polygonaceae )
• 3.Tobacco – (Nicotiana tobaccum – Family –
Solanaceae )
• 4. Ruta – (Ruta graveolens – Family – Rutaceae )
• 5. Tea – Thea sinensis – Family – Theaceae )
• 6. Eucalyptus macroryncha ( Myrataceae )

43. ISOLATION
• Source -
• Eucalyptus macroryncha ( Myrataceae )
• Boil the powder drug with boiling water .
Filter while hot and collect the filtrate .
Cool for the precipitation of the rutin.
Recrystallize it from boiling water , dry
the product.

44. IDENTIFICATION
Paper chromatography -
Solvent system –
n- butanol- acetic acid – water - 5% acetic
acid
Detection – Visible, UV light
Standard reference – Rutin
Sample – Extract powdered drug with 70%
alcohol
Extract fresh drug with 90%
alcohol

45. ESTIMATION :
• By colorimetry :
• Standard solution – Rutin in alcohol
( 100µg/ml)
• Test sample – weigh sample equivalent to
10mg rutin and add 80ml of ethanol. Boil
on water bath and cool to room
temperature .make up the volume to
100ml with ethanol.

46. • PROCEDURE :
• Take 1,2,3,4,5 and 6ml of solution of pure rutin in
alcohol into test tubes and add alcoholic
aluminium chloride, acetic acid and 1N Hcl adjust
the volume to 25ml with distilled water. Measure
the absorbance of the sample against sample
,blank and standard at 420nm. Calculate the
amount by comparison.

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48. PHYLLANTHIN
• Phyllanthin and Hypophyllanthin are the
two major constituents of the drug
phyllanthus. They are Lignans .
• Chemically , Phyllanthin is a diaryl butane,
where as hypophyllanthin is an aryl tetra-
hydronaphthalein

49. • Lignans are a group of chemical
compounds found in plants .Lignans
are one of the major classes of phyto-
estrogens, which are estrogen like
chemicals and acts as antioxidants.
• The other classes of phyto-estrogens
are the isoflavones, they are
polyphenolic in nature.

50. • Biological source : It consists of the aerial
parts of the plant Phyllanthus amarus
• Family – Euphorbiaceae
• Uses :
• Phyllanthin and Hypophyllanthin are
reported to have Hepatoprotective activity.

51. • The drug (Phyllanthus) exhibits antiviral
activity , particularly on Hepatitis B in
humans
• It exhibits Diuretic and Hypotensive
effects in humans .
• The plant possesses antibacterial and
antifungal activities.

52. ISOLATION
• PROCEDURE :
• 1.Mix the powder drug with lime water , It
is allowed to stand for overnight .
• 2.The mass is then transferred to a
Soxhlet apparatus and extraction is
carried out with petroleum ether.

53. • 3.Collect the petroleum ether extract and
concentrate under reduced pressure.
• 4. Mix the extract with methanol and boil , collect
the dewaxed methanol extract , evaporate to
dryness, collect the residue.
• 5. Dissolve the residue in petroleum ether ,
concentrate and allow to stand (Yellow oil gets
separated)

54. • 6. The residue is subjected to column
chromatography on Alumina and elute with n-
hexane: ethyl acetate (99:1)
• 7. 99- 108 fractions corresponds to
hypophyllanthin and 109- 137 fractions
corresponds to phyllanthin
• Subject them to chromatography further to yield
pure phyllanthin and hypophyllanthin

55. IDENTIFICATION
• BY T.LC :
• Adsorbent – Silica gel GF254
• Solvent system – n- hexane: ethyl acetate
(2:1)
• Detection – Vanillin in sulphuric acid
reagent
• Rf value – Phyllanthin – 0.20 ( Blue spot)
• Hypophyllanthin – 0.25

56. ESTIMATION :
• By HPLC :
• Column - µ- Bondapak-18 ( 3.9mm X
30cm)
• Mobile phase – Methanol – Water (66:
34)
• Flow rate – 1.8ml/Minute
• Detection – UV at 230nm

57. • Standard – Known concentration (0.05- 2µg)
• Sample –
• Macerate 1gm of the drug powder with lime water
at room temperature for 18hrs .Reflux with 30ml
methanol containing 3% KOH for 1hr .cool and
filter .collect the filtrate
• Reflux mark with methanol containing 3% KOH .
Filter and collect the filtrate . Combine the
extracts and concentrate under reduced pressure.
Make up to 50ml.

58. • Sampling –
• Apply 10µl of both standard and sample
solutions
• Determination –
• Note the peak areas corresponding to
phyllanthin and hypophyllanthin in both
standard and samples and calculate their
percentages accordingly.

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60. DIGITOXIN
It is the primary active constituent of
Digitalis purpurea
Family – Scrophulariaceae It is a
cardiac glycoside exert highly specific
and powerful action on cardiac muscle.
They make the heart to function more
efficiently.

61. • Thus they are used therapeutically to
strengthens the weakened heart. as
the aglycone part of these glycosides is
the steroidal moiety, they are also
called steroidal glycosides.

62. ISOLATION
• 1.Macerate powdered drug with water at
45 o C for 4- 5 hrs and collect aqueous
extract.
• 2. Macerate mark with 20% methanol for
24hrs and collect methanol extract.
• 3. Combine aqueous and methanol
extracts

63. • 4.Make alkaline the above extract with
sodium hydroxide solution ( Hydrolysis).
• 5.Extract with chloroform.
• 6.Collect the chloroform extract and
evaporate to dryness.
• 7.Subject the residue to column
chromatography for isolation of digitoxin

64. Synopsis of column chromatography –
 Adsorbent –
 Silica gel G
Solvent system –
Follow gradient technique , initially with carbon
tetrachloride, followed by ethyl acetate and
methanol.

65. • Carbon tetrachloride fraction : Colouring
matter (Pigments)
• Ethyl acetate fraction : Flavones and
anthraquinones
• Methanol fraction : Digitoxin ( Rf value-
0.486)
• Digitoxin is purified by recrystallization
from alcohol and diethyl ether (1:1) mixture

66. IDENTIFICATION
• 1. Legal test – Dissolve the extract in
pyridine and add sodium nitroprusside
solution – Pink to red colouration on
making alkaline.
• 2. Baljet test – Extract with few drops of
sodium picrate solution – Yellow to orange
colour

67. • 3.Keddes test –
• Sample with few drops of 3,5-
dinitrobenzoic acid in methanol and few
drops of potassium hydroxide solution –
Reddish / bluish colour.
• 4. Raymond's test –
• Sample with few drops of dinitrobenzene
and few drops of methanol potassium
hydroxide solution – Bluish voilet colour.

68. ESTIMATION
• By Spectroscopic method
• By Bioassay
• By Spectroscopic method – ( I.P .1966)
• Weigh accurately about 40mg of
digitoxin and dissolve in 100 ml of
ethanol. Dilute 5ml of this solution to
100ml with ethanol. Add 3ml of alkaline
picric acid solution to 5ml of this
diluted solution.

69. • Allow to stand for 30 minutes and
measure its absorbance against blank
at 495nm

70. • By Bioassay method-
• Digitalis and its preparations can be
biologically assayed for their potency.
• Principle –
• Comparison of the effect of a known
dilution of the drug with that of the similar
dilution of standard preparation forms the
basis of bioassay.

71. • Test animals –
• I.P. Suggests the use of both pigeons and
guinea pigs.
• Procedure –
• Adult guinea pig are anaesthetized lightly
with ether. The jugular vein of
immobilized guinea pig is exposed and
cannulated. Definite volumes of diluted
preparations are introduced at 5 minutes
of interval until it dies from cardiac
arrest.

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74. DIOSGENIN
IDENTIFICATION – By thin layer chromatography
Synopsis –
Adsorbent – Silica gel 60
Solvent system – Toluene : Ethyl acetate ( 7:3)
Reference standard – 1mg/ml Diosgenin in chloroform

75. • Preparation sample –
• Reflux the powder with 50ml of 10% hydrochloric acid for
2hrs , collect the residue. Wash the residue with dilute
sodium carbonate solution , collect the residue. Extract the
residue with solvent ether successively , combine the ether
extracts and concentrate. Dissolve the residue in 2ml of
chloroform .

76. • Procedure – Apply 20µl of test and standard solutions
on prepared plate.
• Detecting reagent – Spray with anisaldehyde
sulphuric acid reagent
• Rf value – 0.37 ( Dark green spot)

77. ESTIMATION – BY HPTLC
• Standard preparation –
• Prepare known concentration of solution in chloroform The
amount of substance in the spot is 2- 25µg.
• Sample preparation –
• Reflux 1gm of drug with 2.5N Hcl for 4hrs, cool and filter
through Whatman filter paper , collect the residue and dry at
a temperature less than 80O in an oven . Extract with
petroleum ether in a soxhlet for 4 hrs and evaporate the
petroleum ether extract to dryness.

78. • Dissolve the residue in chloroform , make up the volume
to 10ml with chloroform
• Solvent system – Toluene – Ethyl acetate (7:3)
• Procedure - Apply known volume of standard and sample
preparations in triplicate on precoated HPTLC plates and
develop the plate to a distance of 8 cm.
• Detecting reagent – Liberman- Buchards reagent and
heat at 120o

79. • Cool and scan in a densiometer in reflection mode at
600nm.
• By comparing the areas corresponding to diosgenin in
sample and standard preparations , the amount of
diosgenin in the sample is estimated.

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81. ASIATICOSIDE
• Asiaticoside and medecassoside are the important
triterpenoidal saponins of Mandukaparni ( Brahmi).
• Source : It consists of dried aerial parts preferably
leaves of Centella asiatica.
• Family: Apiaceae (umbelliferae)

82. • Uses :
• Brain tonic
• Nerve tonic
• Sedative
• Spasmolytic
• Anti-anxiety
• Anti-stress
• In Ayurveda it is described as ‘Rasayan’.

83. ISOLATION -
• Percolate the powdered drug with 90% alcohol. Collect
the alcoholic extract and extract successively with
petroleum ether, diethyl ether, ethyl acetate and n-
butanol.
• Collect the n- butanolic extract and concentrate and
the residue is extracted with acetone. Filter while in
hot, filtrate on cooling forms precipitate.

84. • Subject the precipitate for preparative HPLC with
Methanol : water (1:3)
• Isolation of Asiaticoside and Medecassoside

85. • Identification:
Give test for triterpenoidal saponins
• 1) Salkowski test.
• 2) Lieberman Burchard reaction.
• 3) Trichloro acetic acid test.

86. a) Salkaowski test: -
A few drops of concentrated sulphuric acid were added
to the chloroform solution, shaken and allowed to stand.
Lower layer turned yellow.
b) Lieberman Burchardt test: -
To the chloroform solution a few drops of acetic
anhydride and 1ml of concentrated sulphuric acid was
added. A deep red color was produced.

87. c) Trichloro acid and Stannic Chloride test: -
To the chloroform solution a few drops of thionyl
chloride and a pinch of stannic chloride were added. A
range of colors green, blue, purple and finally turning to
red were obtained.

88. • ESTIMATION : By HPLC method.
• Column - µ - Bondapack C 18 (8mm X10 cm)
• Mobile phase - Acetonitrile : water (1:3)
• Flow rate - 1.5 ml/minute
• Detection - UV at 205nm
• Standard - known concentration of Asiaticoside: 0.02 to
0.4 mg/ml in methanol, Madecasosside: 0..2 to 4mg/ml.

89. • Sample preparation –
• 2 gm of the drug , reflux with 90% methanol on water
bath , cool and filter, collect the filtrate, concentrate
under vaccum, make up to 50ml , dilutions can be
made as per the requirements.

90. • Procedure:
Inject 10ul sample and standard. Record the peak
areas of asiaticoside and madecassoside of test and
standard. Accordingly, the percentage of asiaticoside
and madecassoside present in the sample can be
calculated.

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92. • Sennosides are the active constituents of
Senna. They are Sennoside A,B,C and D.
They are dimeric anthraquinone
glycosides.
• Biological source: It consists of dried
leaflets of cassia angustitolia and cassia
acutifolia.
• Family: Leguminosae.

93. • Uses:
• Senna is a purgative drug, it is irritant
purgative due to presence of
Anthraquinone derivatives.

94. Methods of Isolation:
Various methods are available for isolation of
sennosides. But essentially, they are isolated as
calcium sennosides because of better stability.
The various methods are:
1.Extraction of Sennosides as their Calcium salts
2.Ahmed and Samia method
3. Stoll and samia method,

95. 4. Modification of stoll and Becker method
5. Notherman and lick method
6. Mauzaram et.al method.

96. EXTRACTION – AS CALCIUM SENNOSIDES
• 1. Extract the powder drug with benzene
for 2hrs on an electric shaker.
• 2. Filter and collect the marc. And dry the
marc.
• 3.Extract the dried marc with 70%
methanol for 4hrs and collect the
methanol extract .

97. • 4. Re- extract the marc with methanol.
• 5. Combine the above( 3 & 4) extracts and
reduce the volume to 1/8 of original volume .
• 6. Acidify the concentrated extract to pH 3.2
with dilute hydrochloric acid and set aside for
2hrs at 50 o C .
• 7. Filter and add anhydrous calcium chloride in
spirit to the filtrate.

98. • 8. Adjust the pH to 8 with the help of
ammonia and set aside for 2hrs.
• 9.Finally collect and dry the precipitate
(Calcium sennosides)

99. IDENTIFICATION -
• Borntragers test –
• Boil powdered leaves with dilute
sulphuric acid . Filter and collect the
filtrate. Cool it.
• Now shake with an organic solvent like
benzene /chloroform. Separate the
organic layer and add equal quantity of
ammonia

100. • Ammonia layer becomes pink or red
indicating the presence of anthraquinone
glycosides.
• By Thin layer chromatography –
• Adsorbent – Silica gel 60F254
• Solvent system – n- propanol: ethyl
acetate: water (4:4;3)

101. • Detection – Nitric acid in potassium
hydroxide reagent or visible / UV- 366
• Test sample –
• Extract 1gm of powdered drug with 5ml
methanol by heating on a water bath
for 15minutes . Filter and use filtrate
as sample.

102. • Visualization –
• UV 366nm – Lemon yellow or Light blue.
• Rf values – Sennoside A –0.4
• Sennoside B - 0.2
• Sennoside C - 0.7
• Sennoside D - 0.5

103. ESTIMATION -
• It is estimated by Calorimetrically ( IP
1996)
• 1. The drug is powdered and extracted
with water or hydro- alcoholic solution.
The aqueous phase is extracted with
chloroform or ether ( Eliminates free
anthraquinones)
• 2. The aqueous solution is oxidized with
Ferric chloride and hydrolyzed with Hcl

104. • 3. The resulting anthraquinones are
extracted with organic solvent chloroform
or ether. The solvent is evaporated and
the residue is redissolved in a
methanolic solution of magnesium
acetate, whose absorbance is measured
at 515nm (Red colour)
• Standard solution–1:8 dihydroxy
anthraquinone

105. • By Bioassay –
• The bioassay is carried out on mice or
rats by counting the number of wet
faecus after administration of the
drug according to the weight of animal
for determining the activity.