King Saud UniversityPharmacy College

Pharmacognosy Department

322PHGPractical course

TA/ Hanan AL- Ati & Ghadah AL-Hmoud

PHG

322

322 PHG Lab. syllabus: (2nd. semester 1432/1433)

SubjectsDateLab#

Introduction of alkaloid + Assay of Nicotine

13,15,16/3/14331

Assay of Reserpine 20,22,23/3/14332

Assay of Tropane Alkaloids(Strychnine in Nux vomica)

27,29,30/3/1433

3

Qualitative Identification of Alkaloids (part I)

4,6,7/4/14334

Qualitative Identification of Alkaloids (part II)

11,13,14/4/14335

Microscopical analysis of powder drugs containing Alkaloids + revision

18/20/21/4/14336

Practical Exam + Quiz25,27,28/4/14337

Introduction of glycosides +Assay of senna

9,11,12/5/14338

Assay of Cardiac Glycosides 16,18,19/5/1433

9

UV Spectrum of Flavanoid 23,25,26/5/1433

10

Qualitative Identification of Glycoside

1,3,4/6/143311

Microscopical analysis of powder drugs containing glycosides

8,10,11/6/143312

Revision15,17,18/6/143313

Practical Exam + Quiz22,24,25/6/143314

Safety:

Porcelain dish

Round bottom flask

Lab#1

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322

Drug abuse(Assay of

nicotine in urine.)

Pharmacognosy Department

Drug abuse:Drug abuse is related to taking

a psychoactive drug or performance enhancing drug for a non-therapeutic or non-medical effect.

Abuse drug can be classified according to it's effect on CNS into:1. CNS depressants

a. Narcotic analgesics (Opioid): e.g. Morphine

b. Sedatives and hypnotics: e.g. Barbiturates

2. CNS stimulants

a. Caffeine and nicotine

b. Cocaine

3. Hallucinogens

a. LSD

Nicotine.• Nicotine, is a liquid alkaloid which exists chiefly as

malate in the leaves of Nicotiana tabacum.

• In low concentrations (an average cigarette yields about 1 mg of absorbed nicotine).

• Nicotine first stimulates nerve cells and then paralyses them.

• The primary therapeutic use of nicotine is in treating nicotine dependence in order to eliminate smoking (gums, dermal patches and lozenges).

AlkaloidsWhat is an ALK.?

Is nitrogenous comp. having complex molecular structure and significant pharmacological activity, most of them is basic in nature, most from plants.

Assay of Nicotine in urine.

Principle:

Procedure:

nicotine

r blu

Crimsom green to

blue

Lab#

Student name: ID:

E.P=

Calculation:

Lab#2

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322

Assay of Reserpine.

Structure: Indole alkaloids.

Uses: As hypotensive agent

Procedure:

Assay of Reserpine.

Pharmacognosy Department

Comments on the assay : 1-Citric a`inc. the solubility of reserpine & prevents its hydrolysis.

2-Reserpine could be extracted from acidic medium by CHCl₃ since it is weak base.

3-Sod. Hydrogen carbonate is used to remove excess a`.

6- H₂SO₄+sod. nitrite Nitrous a` which react e` the alkaloid & give the yellow colour.

7- Sulfomic a` is used to decompose excess nitrous a`.

Lab# Student name: ID:

E.P=

Calculation:

Lab#3

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Assay of liq. Extract of Nux

Nux vomica: - bitter-tasting drug obtained from the poisonous seeds of Strychnos nux-vomica.

- The dried seeds contain strychnine and brucine, both colorless crystalline alkaloids, as well as sugar, acid, and oil.

-I n the past nux vomica was used as a tonic in the form of a tincture, or alcoholic solution, but it is not used in modern medicine. - In minute quantities it has a powerful peristaltic action on the intestines and in larger doses causes convulsions and death.

Principle:

Pharmacognosy Department

Procedure )3 steps(:

(1) Purification:

(2) Alkalinization:

Red blue

pipette

(3) Extraction:

Comments on the assay: 1) To insure that the alka. Is in salt form.

2) To remove impurities.

3) To remove any volatile bases & the CHCl₃ remain.

4) To dissolve any resonance from the alkaloid bec. it will decrease the reading.

Lab# Student name: ID:

E.P=

Calculation:

Lab#4 & 5

PHG

322 Qualitative Identification of

Alkaloids(part I&II)

Pharmacognosy Department

Chemical test for qualtitive identification of Alk. :

3 (Marqui’s reagent:Formaldehyde+ conc. H₂SO₄

Specific Alk. colour reactions.

1)Ephedrine:

2)Brucine:

3)Strychnine:

)20%(NaOH

u

Lines radiating from crystals

4)Atropine:

5) Papaverine:

ORANGE/RED

Fades rapidely

6) Caffeine:

7) Quinine:

BROWN PPT.

Brown ppt.

Scheme for Unkown AlK. Sol.

emerald

Lab#6

PHG

322Microscopical analysis of powdered drugs containing alkaloids

A( Belladonna leaves:

• Microscopical examination:a) Idioblast

containing crystals of Ca Oxalate (Microsphenoidalcrystals)

b) Glandular hair (multicellular stalk

Pharmacognosy Department

Atropa belladonna

& unicellular heads)

c) Anisocytic Stomata

• Active constituents: 1- Hyoscyamine, hyoscine & atropine2- Volatile base.

• Uses: act as local anesthetic & as sedative to relieve spasmodic cough.

B( Datura leaves:

Alkaloids

Datura stramonium

• Microscopical examination:a) Crystal layer

of Cluster crystal of Ca Oxalate

b) Non Glandular hair(multicellular,unicerate)

c) Anisocytic Stomata

d) Glandular hair (unicellular stalk & multicellular head)

• Active constituents: 1- Contain mainly hyoscyamine & hyoscine Atropine may be formed

from racemisation of hyoscyamine

• Uses:1- In motion sickness due to sedative action of hyoscine.2- Dilate the eye pupil due to atropine presence.

C( Hyoscyamus leaves:

• Microscopical examination:a) Ca Oxalate

(Twin prism)

Alkaloids

Green

Hyoscyamus niger

b) Branched Hair (Glandular hair)

c) Anisocytic Stomata

• Active constituents: 1-Hyoscyamine,hyoscine & atropine.

• Uses:Relieve the spasm, as sedative & in insomnia.

D( Cinchona bark:

Alkaloids

Cinchona succirubra

Rubiaceae

• Microscopical examination:a) Phloem fibers

e`funnel shaped lumen

b) Parenchyma cells containing Ca oxalate

c) Starch granules

d) Cork cell

• Active constituents: 1- Quinine2- Quinidine3- Cinchonine4- Cinchonidine

• Uses:Stomachic, antimalarial due to the presence of quinine.

Alkaloids combined with tannin (cinchotannic acid)

• Chemical tests:

Cinchona pd. Red fume

E( Ipecacuanha root:

• Microscopical examination:a) Acicular

crystals of Ca oxalate

b) scleride

c) Starch granules

d) Cork cells

flame

Cephaelis ipecacuanha

• Active constituents: 1- Isoquinoline alkaloids: emetine, cephaline

• Uses:1- expectorant and emetic2- ameobic dysentry due to presence of emetine

Lab#7

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1St. Practical Exam.

Pharmacognosy Department

Lab# 8

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Assay of Senna

Pharmacognosy Department

Glycosides

What are Glycosides?They are gp. of plant constituents which upon hydrolysis yield sugar & non sugar parts.

Glycosides

sugar part )glycone( Non sugar part )aglycone(

stability & solubility pharmacological activity

Link b/t sugar & non sugar part gives the following types of glycosides :

C-glycoside

O-glycoside

N-glycoside

S-glycoside

e.g, Senna leaf

Function in the plant:

1- Energy producer during seedlings through hydrolysis of steroid compounds.

2- Detoxifying agents for many harmful substances that are transferred to the barks, seed coats & fruits in the form of glycosides.

3- Attractive substance.4- Protective against microorganism & insects.

10

10-

10-10-

Classification of glycosides :

1) According to the chemical nature of aglycone:

e.g. Senna anthraquinone glycoside. Steroid steroidal glycoside.

2) According to the active constituents:

e.g.Cyanogenetic glycosides containing (HCN) e.g.Amygdaline.

3) According to the therapeutic activity:

e.g.Cardiac glycosides e.g.Digitalis.

4) According to the plant origin:

e.g.salicin from Salix species.

Types of glycosides :

1( Primary glycosides:The unchanged form of glycoside which occur naturally in the fresh plant & not the product of any stage of hydrolysis of a glycoside.

2( secondary glycosides: Result from the hydrolysis of 1ry glycosides,usually by (enzymatic,acidic & alkaline) activity e.g. of enzymes: Emulsin enz. & Myrosin enz.

Examples of glycosides:

1- Phenolic glycoside. e.g. Salicin2- Anthraquinone glycoside. e.g. Senna, Aloe…….etc3- Cardiac glycoside. e.g. Digitalis.4- Flavonoid glycoside. e.g.Rutin5- Tannin.e.g. Tea & gall.6- Saponins. e.g.Quillaia bark.

Assay of Senna

Senna

Free anthraquinone. Combined anthraquinone.

Sennidin (aglycone) Sennosides (glycoside)

Main anthracin in Senna is4 types of sennoside (A,B,C,D).A=optically active (levo rotatry) B=Optically inactive (meso rotatry)A&B both have COOH. C & D both have CH2OH.

Glycoside = Soluble in polar organic solvents. Aglycone = Soluble in Organic solvents insol.in In H2O.

Sugar = Soluble in H2O insol. in organic solvents .

Principle :

Procedure:

On W.B.

Shake

Rose red colour

Lab# Student name: ID:

E.P=

Calculation:

Lab#9

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Pharmacognosy Department

Spectrophotometric assay of cardiac glycoside in Digoxin By 4 methods:

1) Xanthydrol method.

2) Digoxin reagent method.

3) Baljet reagent method (Picric a`:NaOH).

4) Kedd`s reagent method (dinitrobenzoic a`: NaOH)

Note:

1) Method no.(1&2) are used to determin the deoxy sugar part while (3&4) are used for aglycon part.

2) These methods are stability indicated in the solid form. Bec. solids are more stable than liquids. since in sol. form aglycone results from hydrolysis during storage w`may effect in abs. e`sugar present in glycoside.

So what we can do? (principle)

Procedure: )U.S.P method )

Lab# Student name: ID:

E.P=

Calculation:

Lab#10

Importance of U.V. spectrum of flavonoids:

PHG

322U.V. spectrum of flavonoids

Pharmacognosy Department

1(For determination of unknown by compression e`standard.

2(For determination of no. & position of OH gp. by specific reagents.

Structure of Flavonoids:

Note:Cinnamoyl part have U.V. abs. higher than benzoyl part bec. It has conjugations w`make resonance that make high abs.

e`MeOH: Flavone (aglycone) = BandI (304-346)

(glycoside) Flavonol (aglycone) = Band I (352-385) (glycoside) = Band I (346-365)

Shift reagents:

The use of shift reagents for identification of free OH in flavones & flavonols.

A-BandI:1( NaOMe: -(sp.for 4´-OH) . - give bathochromic shift)40-60nm(.

-make ionization for OH gp & make conjugation.

2( AlCl3:

3( Boric a`)H3BO3(: orthodihydroxy system e`OH at C3`,C4`or C4`,C5` or both (a`labaile) (bathochromic shift in band I )12-35nm(.

B) BandII: 4( Na acetate:

OH at C7 (bathochromic shift in bandI I) )5-20nm(.

Interpret the spectrum data and draw the structure of the compound.

Compound (A): (Molish , s test -ve)

MeOH 240, 366 MeOH +NaOMe 236, 420

MeOH +AlCl3 290, 401 MeOH +AlCl3 + HCl 291, 401 MeOH +NaOAc 266, 320

MeOH +NaOAc +H3BO3 208, 364

Interpret the following data and draw the structure of the compound.

EXCERSISES

Compound (A), Molish,s test +ve

Yellow compounds show the following UV. Spectral data : (λmax (nm))

Compound NO. (1): (Molish , s test –ve)

MeOH: 252, 307NaOMe: 237, 380AlCl3 290, 394AlCl3 + HCl: 291, 394NaOAc: 266, 400NaOAc +H3BO3: 208,300

Compound NO. (2): (Molish , s test –ve) MeOH: 247, 341NaOMe: 293, 400AlCl3: 258, 380AlCl3 + HCl: 247, 341NaOAc: 277, 400NaOAc +H3BO3: 257,365

Interpret the above spectral data and draw the structure of the compounds.

Lab#11

Examples of glycosides:

PHG

322

Qualitative Identification of

Glycosides

Pharmacognosy Department

1(Phenolic glycosides:

2 )Anthraquinone glycosides: )senna leaves(

( water extract )

b)Aloe (alc. extract)

3(Cardiac glycosides: )Digitalis pd.( )alc.ext.(

Cardiac glycosides

Cardenolides )5-membered ring( Bufadienolides )6-membered ring(

d) Lieberman-Burchard test(specific for steroid nucleus):

purple

Orange-red

Cardenolides

4(Flavonoids glycosides: )Rutin( ) alc.ext.(

5( Tannins: )H2O. extract(

6(Saponins: )Quillaia bark, liquorice root(

Effervescence & evolution of red fumes

Lab#12

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Microscopical analysis of

powdered drugs containing

Pharmacognosy Department

1( Senna Leaves:

• Microscopical examination:a) Paracytic

stomata

b) Ca Oxalate: Crystal Sheath

c)Simple hair (unicellular uniglandular hair covered by warty cuticle)

• Active constituents:1- Sennosides A,B,C&D 2- Aloe-emodin & rhein in small amount.

• Uses: As purgatives especially in cases of habitual constipation, in small dose, it is used as laxative.

2(Digitalis leaves:

• Microscopical examination:a) Collapsed hair

(multicellular,non glandular)

b) Unicellular stalk, Multicellular head)

Anthraquinone glycosides.

(multicellular stalk,unicellular head)

a-From below,b-from the side,c-fromabove

c) Anomocytic Stomata

• Active constituents: 1-Purpurea glycosides A & B.

2-Hydrolytic enzyme & oxidase which form secondary glycosides (digitoxin & gitoxin)

• Uses:As cardiotonic in most forms of cardiac failure.

3( Squill bulb:

Primary glycosides.

• Microscopical examination:a) Raphides

crystals of Ca Oxalate (bundle,free,broken)

b) Epidermal cell

• Active constituents: - crystalline glycosides scillaren A

- an amorphous mixture scillaren B

• Uses:heart tonic, in small doses as expectorant but in large doses as emetic.

Cardiac glycoside

4( liquorice root:

• Microscopical examination:a) Starch

granules

b) Ca oxalate (prisms)

c) Fibres with in complete Ca oxalate prism sheath

• Active constituents: a- sweet principle glycyrrhizinb- flavonoids (liquiritin, isoliquiritin)

c- coumarin

• Uses:1- mild expectorant

2- gastric ulcer

3- rheumatoid arthritis

• Chemical test : 1- powdered liquorice + 1-2 dps of H2SO4 orange color

2- Shake with water permanent froth (10-15 min.)

Lab#13

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Revision

Lab#14

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Final Practical Exam

Pharmacognosy Department

Pharmacognosy Department