PHYT

OPHARMACEUTI

CALS

– QUIN

INE

DR.U.S

RINIV

ASA

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

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.

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.

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. 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 oC.

IDENTIFICATION

1. Chemical tests

2. Thin layer chromatography

T.L.C:

Adsorbent : Silica gel -G

Mobile phase – Chloroform –

diethylamine (9:1)

Detection – Spray with methanolic KOH

; Sulphuric acid reagent or UV Light

PHYT

OPHARMACEUTI

CAL –

EPHEDRIN

E

DR.U.S

RINIV

ASA

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.

• 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.

USES :

Ephedrine is a bronchodilator and

useful in the treatment of asthma and

allergic conditions such as rhinitis,

fever etc.

Used as mydriatic

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.

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)

• 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.

• 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.

• 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

• ESTIMATION :

• 1 . Titration methods :

• A. Back titration method

• B. Non –aqueous titration method

• 2. Colorimetry

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.

• 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.

• 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

• 3. By Colorimetry :

• PRINCIPLE :

• Being a secondary amine , ephedrine

forms a colored complex (400nm )

with Picryl chloride in benzene

• 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.

• Place them in a water bath at 75-77oC 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.

• 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 75-77oC for 20 min. and then allowing

to stand for 3 min at room temperature.

• Calculation:

• The concentration of the ephedrine

in the sample is obtained by

interpolation of the standard curve

PHYT

OPHARMACEUTI

CAL –

ANDROGRAPHOLID

E

DR.U.S

RINIV

ASA, M.P

HARM,P

H.D

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

• Biological source : Kalmegh consists

of dried leaves of the plant known

as Andrographis paniculata

• Family : Acanthaceae

• Uses : Hepatoprotective , bitter

tonic

ISOLATION

• PRINCIPLE :

• Isolation is based on solubility of

andrographolide, it is soluble in

methanol and ethanol

PROCEDURE

• 1.Extract the dried coarse powder

successively with petroleum ether

(60-80oC) ,chloroform and methanol

• 2. Collect the methanol extract and

concentrate and treat his with

charcoal for 24hrs

• 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.

• 5.Purify the combined crystals from

methanol to get andrographolide

( M.P 228-229 oC )

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)

• Reference standard-

Andrographolide 1mg in 1.5ml of

methanol

• Test sample – Extract in methanol

• Detecting reagent –

• 20% sulphuric acid in methanol, heat

at 120oC for 10 minutes

• Rf value of Andrographolide – 0.70,

brown color spot.

• ESTIMATION METHODS :

• 1.Gravimetric method

• 2.Colorimetric method

• 3.High Performance liquid

chromatography (HPLC)

• 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

• 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 concentrations of

andrographolide (0.5- 10µg/ml )

• 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.

PHYT

OPHARMACEUTI

CAL –

RUTI

N

DR.U.S

RINIV

ASA, M.P

HARMA, P

H.D

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.

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 )

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.

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

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.

• 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.

PHYT

OPHARMACEUTI

CAL –

PHYL

LANTH

IN

DR.U.S

RINIV

ASA, M.P

HARMA.P

H.D

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

• 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.

• 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.

• 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.

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.

• 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)

• 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

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 ( Brown spot)

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

• 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.

• 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.

PHYT

OPHARMACEUTI

CAL -

DIGIT

OXIN

DR.U.S

RINIV

ASA, M.P

HARMA,P

HD

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.

• 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.

ISOLATION

• 1.Macerate powdered drug with water at

45oC 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

• 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

Synopsis of column chromatography –

Adsorbent –

Silica gel G

Solvent system –

Follow gradient technique , initially with

carbon tetrachloride, followed by ethyl

acetate and methanol.

• 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

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

• 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.

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.

• Allow to stand for 30 minutes and

measure its absorbance against

blank at 495nm

• 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.

• 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.

PHYTOPHARM

ACEUTI

C

AL - D

IOSGEN

IN

DR.U.SRINIVASA,D.PHARM, M.PHARMA,M

PHIL,PH.D

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

• 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 .

• 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)

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.

• 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

• 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.

PHYT

OPHARMACEUTI

CAL –

ASIATI

COSIDE

DR.U.SRINIVASA,

D.PHARM,M.PHARM,M

.PHIL,PH.D

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)

• Uses :

• Brain tonic

• Nerve tonic

• Sedative

• Spasmolytic

• Anti-anxiety

• Anti-stress

• In Ayurveda it is described as ‘Rasayan’.

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.

• Subject the precipitate for preparative HPLC

with Methanol : water (1:3)

• Isolation of Asiaticoside and Medecassoside

• Identification:

Give test for triterpenoidal saponins

• 1) Salkowski test.

• 2) Lieberman Burchard reaction.

• 3) Trichloro acetic acid test.

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.

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.

• 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.

• 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.

• 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.

PHYT

OPHARMACEUTI

CAL –

SENNOSIDES

DR.U.S

RINIV

ASA, MPH

ARM,PHD

• 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.

• Uses:

• Senna is a purgative drug, it is

irritant purgative due to presence of

Anthraquinone derivatives.

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,

4. Modification of stoll and Becker

method

5. Notherman and lick method

6. Mauzaram et.al method.

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 .

• 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 oC .

• 7. Filter and add anhydrous calcium chloride in

spirit to the filtrate.

• 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)

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

• 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)

• 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.

• 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

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

• 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

• 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.