for sem 5 & 8 as per GTU Syllabus

1. Phytochemical Screening
of Herbal drug
Prepared by :
Pooja Khanpara
APIP
Jamnagar

2. Glycosides

5. Category – Anthraquinone glycoside
•They possess anthracene or their derivatives as aglycone
•Hydrolysis of these glycoside yields aglycone which are
di, tri, or tetra hydroxy anthraquinone
Introduction
O
O
1
2
3
45
6
7
8
9
10
O
H H
1
2
3
45
6
7
8
9
10
OH
H
O
H OH
1
2
3
45
6
7
8
9
10
Anthraquinone
Oxanthrone
Anthrone Anthranol
4H
2H
Kratika Daniel (Ph.D)

6. Types of anthraquinone glycoside
1. O-glycosides where the aglycone moiety is 1,8
dihydroxyanthraquinone derivatives, e.g.,
O OH
CH2OH
O
O
1
2
45
9
10
Gl
8
O OH
COOH
O
O
1
2
45
9
10
Gl
8
O OH
CH3
O
O
1
2
45
9
10
Gl
8
Aloe-emodin-8-glycoside Rhein-8-glycoside Chrysophanol-8-glycoside
2. O-glycoside where the aglycone moiety partially reduced
1,8 dihydroxy anthraquinone, e.g., Oxanthrone-type.
OH OH
O
H O
2
3
45
6
7 9
10
Gl
8 1
Emodin-oxanthrone-9-glucoside Kratika Daniel (Ph.D)

7. 3. C-glycoside where the aglycone structure (anthrone der.)
OH
CH2OH
OH
O
H C6H11O5
2
3
5
6
7 9
10 4
8 1
Barbaloin
4. O-glycosides where the aglycone moiety is di-anthrone der. (i.e.,
dimmer) e.g., Sennosides where there is C-C bridge between the
anthranol units. Sennoside A&B
O O
H
COOH
OH
O OH
COOH
O
H
2
3
5
6
7 9
10 4
8 1
Gl
Gl Kratika Daniel (Ph.D)

8. SOURCE
•Consists of the dried leaflet of Alexandrian or Khartoum
senna, Cassia senna (C.acutifolia), Tinnevelly senna
(C.angustifolia).
Constituents: - Dimeric anthracene glycosides derived
from two anthrones moieties which may be:
OH OH
CH2OH
O
1
2
45
9
10
8
Aloe-emodin anthrone
OH OH
COOH
O
1
2
45
9
10
8
Rhein anthrone
Kratika Daniel (Ph.D)

9. 1.Similar anthrone moiety (Homo-dianthrones) i.e., 2 rhein anthrone
moieties condensate through two C-10 atomes. Thus it can be exist in
two optical forms, Sennoside A (L- form) & Sennoside B (meso form).
O O
H
COOH
OH
O OH
COOH
O
H
2
3
5
6
7 9
10 4
8 1
Gl
Gl
Sennosides A &B
2. Or different (Hetero-dianthrones) i.e., one rhein-anthrone & one
emodin anthrone, Sennoside C (L- form) and Sennoside D (meso form).
O O
H
COOH
OH
O OH
CH2OH
O
H
2
3
5
6
7 9
10 4
8 1
Gl
Gl
Sennoside C&DKratika Daniel (Ph.D)

12. • 2nd method
• Powder drug add in 75% water in alcohol
• Warmed it. Add 5ml HCl
• Add 100 ml toluene & reflux for 6 hrs.
• Cool it, filter it, separate both layer
• Aq. Layer washed with tolune
• Combine toluene layer & add 10% aq. Sodium hydrogen carbonate
(till pink color)
• A. layer acidified with HCl & ppts. Add ethyl acetate

13. • Ethyl acetate layer evaporated & product recrystalized from
glacial acetic acid.
• A dark yellow compound obtained.

14. Spraying reagent:
Nitric acid in potassium hydroxide reagent
Test sample –
• Extract 1gm of powdered drug with 5ml methanol by heating on a
water bath for 15minutes .
•Filter and use filtrate as sample.
H2SO4
Modified BD test: HCl & FeCl3

15. 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

16. • ESTIMATION –
• It is estimated by Calorimetrically ( IP1996)
• 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
• 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

18. Methi/ Fenugreek
B.S.: It obtain from the plant Trigonella foenumgroecum Linn.
Family: Leguminosae
• Afghanistan, Pakistan, India, Iran, Nepal, Bangladesh, Argentina,
Egypt, France, Spain, Turkey, and Morocco.
• The largest producer is India.
Rajasthan, Gujarat, Uttarakhand, U.P., M.P., Maharashtra, Haryana,
and Punjab. Rajasthan accounts for over 80% of India's output.

19. 4 method for isolation
• 1- Alcoholic extraction
• 2- Acid hydrolysis
• 3- Fermentation cum Acid hydrolysis
• 4- Incubation cum Acid hydrolysis

20. 1. Alcoholic Extraction Method
Plant cut into small pieces & dried under the sun
Powder is ext. with methanol/ethanol for 6-8 hrs.(2times)
Filter it concentrate it.(syrupy liquid)
Conc. Liq. Is then hydrolyzed using HCl/H2SO4 for 2-12 hrs.
85% diosgenin is ppts.
Filter & wash with water.
Purification with alcohol

21. 2. Acid Hydrolysis method
Pow.drug first hydrolysis by refluxing with 5% HCl for 2 hrs.
Filter it & washed with H2O & 5%NaHCO3 (2 times)
Finally washed with H2O & make it neutral
Residue further ext. with toluene for 8 hrs.
Filter it.
Conc. it, & get diosgenin ppts.
Filter it.
Wash with little hexane & dried (40-60 °C) to get 95% pure
product

22. 3. Fermentation cum Acid hydrolysis
Fresh green roots collected & smashed in a hammer mill.
The mesh is placed in the fermentation bin & allowed for
fermentation for 2 days
Fermented mesh is dried in sun to reduce moisture upto 7-8 %
Subected to hydrolysis with mineral acid at reduced temp.
Resulting sol. Ext. with heptane to get diosgenin.

23. 4. Incubation with Acid Hydrolysis
Fresh plant material incubated in H2O at 37°C for few days
Then subjected to acid hydrolysis
The concentrated it & with hydrocarbon solvent to get diosgenin.

24. Identification test
1) Libermann Bruchard test:
• Glycoside in acetic anhydride + Few drops of conc. H2SO4
Reddish violet Green
2) Salkovaski test:
drug chloroform sol. + conc. H2SO4 chloroform layer produce
Red color
3. Sulphur powder test:
• Add sulphur powder to the test solution. If it sinks at the bottom the
presene of steroid is confirmed.

25. IDENTIFICATION – By TLC
• Adsorbent – Silica gel 60
• Solvent system – Toluene : Ethyl acetate ( 7:3)
• Reference standard – 1mg/ml Diosgenin in chloroform
• Preparation sample –
• Reflux the powder with 50ml of 10% HCl for 2hrs , collect the
residue. Wash the residue with dilute NaCO3 solution , collect
the residue.
• Extract the residue with solvent ether successively , combine
the ether extracts and concentrate.
• Dissolve the residue in 2ml of CHCl3.

26. • 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)

27. ESTIMATION – BY HPTLC
• Standard preparation –
• Prepare known concentration of solution in CHCl3. 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 80°C in an oven .
• Extract with petroleum ether in a soxhlet for 4 hrs and
evaporate the petroleum ether extract to dryness.

28. • 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
120°
• Cool and scan in a densitometer 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.

42. HPTLC Method

43. Rutin
• It is a bioflavonoid
• Pure rutin is yellow or yellow green color, needle shaped crystal
• Take 20 gm powder soxhlet with 250 ml 80% ethanol.
Filter it, mix it with 25 ml water &
extracted with pet. Ether & CHCl3
Take Aq. Layer keep in cold for 72 hrs.
Yellow ppts. Seperated.
washed with CHCl3: ethyl acetate: ethanol (50:25:25)

44. Ppts dissolved in hot methanol & filterit
The filtrate is evaporated to dryness
Get yellow powder (rutin)
Identification Test:
1) With FeCl3 ---- give dark green color
2) With lead acetate ---- orange yellow ppts
3) With ammonium molybdate & antimony trichloride ---- orange
yellow ppts

45. Estimation
1) TLC & Paper Chromatography:
• Precoated aluminum sheet with silica gel G
• Mobile Phase: Ethyl acetate: butanone: formic Acid:
water(50:30:10:10)
• Ethyl acetate: formic Acid: Acetic Acid :water(100:10:11:27)
• In Paper C.: Stationary phase: filter paper (W.-1)
• Mobile Phase: acetic acid: water (15:85)
• isopropyl alcohol: water (60:40)
• 2) Spectrophotometric:
• Disolved in methanol & detect in UV.

49. 1. Thalleioquin Test: sol. of chinchona drug when treated with
Bromin & ammonia it produce emerald green color.
2.

51. Atropine

54. 2nd method
Take Powder drug, ext with 95% alcohol(soxhlet hot percolation)
Ethanolic ext. concentrate it
Add dil. HCl & filter it
extracted with pet. Ether (to remove impurities)
Aq. Sol. Make alkaline with NH3
Extracted with CHCl3 (3 times)
Combine exts. Evaporate chloroform under vaccum
Again ext. with dil. Oxalic Acid
Finally get crystal of Atropine

56. Estimation/Analysis

57. 2) GC/MS:
• 0.5 ml drug sol., 5 µl IS sol. & 1 ml borate sol. (0.1 M, pH 9.3) are
palced in testtube, mix well & pour in to column.
• After 15 min. compound eluted(washing) with 4 ml
Dichloromethan.
• Elute is evaporate to dryness under Nitrogen.
• The residue mixed with 50µl of O-
bis(trimethylsilyl)trifluroacetamide /
Trimethylchlorosilane(BSTFA/TMCS) in 99:1 ratio & warmed at
45° C. for 20 min.
• The resulting sol. Mixed with 100 µl dochloromethan
• Then injected in GC/MS for analysis.

59. Isolation of Reserpine
Roots are powdered & moisten with 10 % NaHCo3 & ext. with
benzene untill give positive reaction with HgI2
Conc. It & add ether & dil. HCl again conc. It & separate acid layer.
Again washed with ether. Make it alkaline with NH3
Then ext. with CHCl3
The CHCl3 ext. washed with 10% Na2CO3
Dry it & purify it by using methanol
Get pure crystal of Reserpine

60. Identification test & Analysis
• 1) Sol. + Vanillin in acetic acid = violet red color
• 2) by colorimetry: Acidic sol. of drug + sodium nitrite
• Analysis: HPLC
• Mobile Phase: sol A (water & sol. B (acetonitrile)
• Both solvents filter with PTFE(polytetrafluroethylene) 0.45
µm membrane.
• Flow rate of mobile phase is 1ml/1min.
• Injection volume: 20µl at 25°C
• UV detection: 268 nm.

62. Isolation of Morphine
CaCl2
•Ext. with CHCl3
•Make it acidic
•Produce ppt. with addition of Ammonia
•Purify morphine with alcohol

63. Identification test

64. Estimation/Analysis
• 1) GC & GC-MS Analysis
• 2) HPLC Analysis
• 3) Ion-Exchange Chromatography

65. Ion-Exchange Chromatography
• It is excellent bcz these alkoloid are bulky & their ability to
ionpair is strong.
• Stationary Phase: amino silica gel (reverse Ion-pair) &
• C18 (Ion-pair)
• Solvent system: H2O/MeoH or H2O/MeCN buffered with
phosphates gives excellent seperation (reverse Ion-pair)
• & H2O/MeoH/AcOH/Sodium heptane sulphonate. (Ion-pair)
• When C18 used: elution sequence as follows
• morphine < codeine < thebaine < noscapine < papavarine

67. Isolation of Ephedrine
Powder drug , moist with Na2CO3 & ext. with Benzene
Filter it
Take residue
ext. With Dil. HCl
Make alkaline by using K2CO3 & add CHCl3
Add Na2SO4 in CHCl3 solution & dried it
Take residue, treat with Oxalic Acid ,warm, filter & cool it
Get Ephedrine oxalate crystal

68. Identification test
• Ephedrine in H2O + dil. HCl treated with  CuSO4 + NaOH
 violate color  add ether  purple color  Aq. Layer
shows blue color

71. Isolation of Caffeine
• Ext. 50 gm pwd. Tea with 200 ml of ethanol for 6 hrs. (soxhlet
app)
• Transfer to ext. in porcelain dish containing MgO suspended in
100 ml of water
• Mixture is evaporated to dryness on waterbath
• In residue add water & make paste. Add more water & make
suspension. & filter & collect the residue.
• In this filtrate add 10% H2SO4 boil it for 30 min.
• While hot, add 25 ml CHCl3
• Add NaOH/KOH in it.(for decolorization)
• Add equal volume of water. Separate CHCl3 layer.
• Evaporat to dry. Add water for purification
• Finnaly get needle shape crystals.

72. • Murexide test:
• In petridish add drug + HCl + KCl  heated & make it dry 
shows purple color vapours of dilute NH4
• It produce ppts. With tannic acid solution.
• TLC:
• Ethyl acetate: methanol: Acetic acid (80:10:10)

73. Resin

75. Crude ext. evaporated, after 3 days add 100 ml toluene
Transfer in seperating funnel
Add 100 ml NaOH o.2M , shaken for few min.
Aqueous Phase collected
In that add HCl (acidified pH 3) (in this step brown ext turns
yellow)
The foltrate is ext. with diethyl ether (300 ml)(bright yellow)
Etharal phase washed with 30 ml water & dried over MgSO4
Finally get yellow color crude curcumin
(purified bt TLC)

77. Podophyllum
Indian Podophyllum
• Synonym – Indian May apple, Wild lemon, Duck’s Foot,
Hog Apple
• Biological source –
It consists of the dried rhizome and root of Podophyllum
hexandrum or Podophyllum emodi
• Family – Berberidaceae
• G.S.: Kashmir to Sikkim in India. Grow in Tibet &
Afghanistan

78. Isolation of Podophyllotoxin
120 gm root powder ext with methanol(300ml)(soxhlet) for 6 hrs.
Take Filtrate . Concentrate it . And add 200 ml water containing 2
ml HCl & cooled it.
Allow mixture to stand 2 hrs. below 5°C filter under vaccum.
Wash again. With acidified water.
Dissolve residue in sufficient qty. of hot alcohol (90%).
Filter & get filtrate , evaporate it. Weigh it. get podophyllotoxin

79. Identification test
• Macerate 0.5g of the drug with 10 ml of alcohol & filter + 0.5
ml Copper acetate  brown ppts
• Alcoholic ext. + 5 ml 1N KOH  stiff jelly is produce.
• Analysis:
• HPLC: performed on Thermo Finnigan HPLC machine with
pump system with 966-photodiode detector, at 283nm.
• Satisfactory result get with E.Merck RP-18 column with diod
array detector & auto –injector
• Mobile Phase: methenol:water (60:40) (for 30 min)
• At flow rate 0.8 ml/min.

80. Terpenoids
Isolation of β-Carotenoids (Daucus carota- Umbeliferae)
Dried powder of carrot ext with pet. Ether
Concentrate it.
Treated with CS2 , add alcohol(to colorless remove impurity)
Trt. Mother loquor With more ethanol ppt of crude carotenes
Filtered , dissolved fresh CS2 & removed impurities by adding
alcohol
Recrystalize by pet. Ether.
• The 3 carotenes α, β, γ are seperated by means of
chometography using Ca(OH)2 & pet. Ether as solvent
• Finnaly carotene eluted by ehter methanol solution.

81. Identification test
• Solubility: inoluble in water.
• Color of a sol. of the sample in acetone disappears after
successive addition of a 5% NaNO3 & 0.5 M H2SO4
• Analysis:
• By spectrophotometer
• By HPLC

82. 2. Mentha
• SYNONYM- Peppermint oil, Mint, Pudina,
Brandy mint, Lamb mint.
• B.S:
• It is volatile oil obtained by steam distillation of
fresh flowering tops of plant Mentha Piperita Linn
• Family- Labiatae
• G.S.: Asia, Europe, North America
• Cultivated in india, japan, Germany, France, Itly.

83. Isolation of Menthol
• Done by steam distillation

84. Identification test
• Few drp. Mixed with 5 ml nitric acid & heated on waterbath.
Within 5 min. liquid develops blue color further heating
shows cooper color fluorescence.
• After sometime its become yellow.

85. Analysis
• Take 10 gm drug, add 10 ml of acetic anhydride & 2 gm of sodium
acetate to the flask.
• Reflex for 1 hr.
• After cooling add 30ml water, boil it for 15 min. with continuous
stirring.
• Then transfer in separating funnel.
• The oil layer is washed with water until it become neutral.
• Add 2 gm of sodium sulphate & shake it.
• Allow it for 30 min. & filter it.
• Take both layer in different flask, add 5 ml ethanol &
Phenolphthelin indicator by adding 25 ml KOH-ethanol
solution.(for neutralize it)
• Again reflux for 1 hr. cool it titrated with HCl.

86. • % Menthol = 7.814 x (c-a) {0.021 x (c-b)/U}
A – 0.021 x (c-a)
Where,
“a” & “b” = ml of HCl for oil
“c” = ml of HCl for blank
U = Unacetylated oil
A = Acetylated oil

87. Lemongrass
• SYNONYM- indian melissa oil, fever grass
east indian lemon-grass oil
• B. S.-
• It is volatile oil obtained by distillation of leaves and aerial
parts of plant of Cymbopogon Flexuous or Cymbopogon
Citratus
• Family- Graminae (Poaceae)
• oil should contain NLT 75% of aldehyde
as citral.

88. Isolation of Citral
• It is Acycilc monoterpenoid
• Isolation done by steam distillation
• It contain 90% of citral-a, & 10 % citral –b
• Identificaton test:
• Test sample + alcoholic solution of sudan III was added. red
color
• Test sample + tincture alkane  red color

89. Analysis
• By gas liquid chromatography(model GC-14B) coupled with a
non-polar DB-5 capillary column using Flame Ionisation
Detector.

90. Artemisin
• It is sami-synthetic derivative of grp of drug that possess the
most rapid action of all current drug against Plasmodium
falciparum
• It is isolated from plant Artemisia annua (sweet warmwood)

91. isolation
• Take powder macerate with solvent methanol.
• Performed in magnatic stirrer with speed of 700 rpm for 1 hr.
• This process repeted till methanol layer became colorless.
• Ext. then evaporated using rotavapour vaccum at temp. 40 °C.
untill volume to 100 ml.
• The ext. solu. Was partitioned using 50 ml haxene. Untill a
colorless haxane layer was obtained.
• 1: hexane ext. & 2: methanol ext.
• methanol ext. add 10 ml water & 50 ml ethyl acetate. Partitioned
untill ethyl acetate layer become colorless.
• By this process, ethyl acetate & methanol-water ext. obtained.
• evaporated using rotavapour vaccum at temp. 40 °C.

92. Identification test
• 1 gm powder boil with 10 ml of alcohol & filtered, NaOH heated- red
color
• Analysis:
• 1) IR: 2mg drug mix with 98 mg of KBr which dried for 24 hr. at temp.
105 °C.
• Analysed at : 4000 cm-1 to 400 cm-1.
• 2) UV : 1mg drug mix with 10 ml methanol & analysed λ 200-400 nm.
• 3) TLC : 1 mg dissolved with 5 ml of ethyl acetate.
• Stationary phase: Silica gel 60 F254
• Mobile Phase: Ethyl acetate: hexane (3:97 or 7:93)
• 4) LC – MS : 1 mg drug mixed with 1 ml methanol & injected as 20 μl
& eluted using a methanol: water (9:1). With flow rate 1ml/min.
• C18 column used.

93. Isolation of Vitamin A
• It is a grp of unsaturated nutritional oraganic compound which
include retinol, retinoic acid, caritenoids.
• It is extracted with CHCl3 or Hexane + ethanol followed by
purification.
• Saponifying done by KOH
• Identification test:
• 1 ml CHCl3 sol. Of retinol + 10 ml antimony trichloride  blue
color
• Analysis:
• by spectrophotometer: incubation method(45 C 2 hr) using N2 gas

94. Antibiotics
1) Penicillin
• Penicillin produce by various strains of P.notatum, P.chrysogenum.
• Isolation of Penicillin: Aseptic condition must needed
• The metabolic solution is rapidly cooled at low pH.
• Then solvent extraction done
• 1) Amyl alcohol – pH is reduced to 2-3
• 2) Butyl alcohol – pH 6.4 after adding Aminium sulphate
• Pet. Ether is add to cold extract & shaken with dil. Sodium
bicarbanate sol. (pH 6-7)
• Then rapidally freeze evaporating yield sodium salt of penicillin.
• During all process temp. must be maintained at temp. just abov
freezing point to avoid inactivation.
• For purification, Penicillin salt is passed through asbestose pads
which absorb micro-organisms & pyrogens.

95. Identification
• By TLC:
• Stationary Phase: Silica gel 60 HF
• For spotting: hamilton syringe
• Solvent kept 60-90 min. min.
• Then run TLC plate
• Afterwards compare with standard Penicillin G.
• Analysis:
• By Photoelectric spectrophotometer (at 322mµ in a
Beckman D.U. quarts)
• 2 procedure used:
• 1st procedure: quantities or conc. Is more in solution
• 2nd procedure: quantities or conc. Is less in solution

96. streptomycin
• The mycelium & waste materials were separated from
antibiotic filtrate. (by charcol)
• It is eluted from the adsorbent by means of dilute aq. Or
alcoholic mineral acids.
• That acidic eluate purified by ion exchang resin
• Pure streptomycin isolated either as sulphate or as crystalline
tri-hydrochloride With calcium chloride.
• Aseptic condition must be maintained.
• For getting completely sterile drug: redissolved to give 25%
sol. Which is free from impurities by passing Seitz filter,
freeze dried.(then transfer in to vials)

97. Identification
• 1) Maltol formation:
5 ml sol. + 1 ml of 2N NaOH. keep test tube in boiling
waterbath for 3 min. & cool for 3 min. caramel like color &
butterscotch like flavour
• 2) Phenol reagent:
Cooled alkaline sol. Of streptomycin + 1 ml phenol reagent,
mix well. Let the sol. Stand for 1-2 min. + 3 ml 20% Na2CO3,
& shake vigorously.
read after 10 min. in photoelectric calorimeter with Filter 660.
Analysis: by HPLC