basics of standerdisation by dr.u.srinivasa, professor and head, srinivas college of pharmacy,...

Dr.U.SRINIVASA, M.Pharm, Ph.D

STANDARDIZATION ?

Standardization involves adjusting the

herbal drug preparation to a defined content

of a constituent or a group of substances

with known therapeutic activity by adding

excipients or by mixing herbal drugs or

herbal drug preparations.

Botanical extracts made directly from crude

plant material show substantial variation in

composition, quality, and therapeutic effects.

In general, quality control is based

on three important pharmacopoeial

definitions:

1. Identity: Is the herb the one it should be?

2. Purity: Are there contaminants, e.g., in

the form of other herbs which should not

be there?

3. Content or assay: Is the content of active

constituents within the defined limits?

Identity can be achieved by macro- and

microscopical examinations.

Voucher specimens are reliable

reference sources. Outbreaks of diseases

among plants may result in changes to

the physical appearance of the plant and

lead to incorrect identification. At times

an incorrect botanical quality with

respect to the labeling can be a problem.

Marker substances are chemically

defined constituents of a herbal

drug that are important for the

quality of the finished product.

IdeallyIdeally, the chemical markers

chosen are responsible for the

botanical’s effects in the body.

Purity is closely linked with the safe use

of drugs and deals with factors such ash

values, contaminants (e.g. foreign

matter in the form of other herbs), and

heavy metals.

However, due to the application of

improved analytical methods, modern

purity evaluation also includes microbial

contamination, radioactivity, and

pesticide residues.

Analytical methods such as photometric

analysis, thin layer chromatography

(TLC), high performance liquid

chromatography (HPLC), and gas

chromatography (GC) can be employed

in order to establish the constant

composition of herbal preparations.

Content or assay

Is the most difficult area of quality control

to perform, since in most herbal drugs the

active constituents are not known.

Sometimes markers can be used. In all

other cases, where no active constituent or

marker can be defined for the herbal drug,

the percentage extractable matter with a

solvent may be used as a form of assay, an

approach often seen in pharmacopeias.

Importance of standardization:

Vegetable drugs are invariably inconsistent

in composition. Seasonal changes,

geographical changes, genetic factors,

edaphic factors i.e age of the plant, season

of the harvest, method of drying etc.,

In any system of medicine , the medicines

must be effective and safe.

In some cases, when bioactive ingredient is

not known, assay is not possible. In many

cases, preparations contain complex

heterogeneous mixtures.

Only in few cases, the drug activity is due to a

single compound. Often many active

constituents contribute the activity. Even inert

constituents may influence the bioavailability

and elimination of active constituents.

Dr.U.Srinivasa, M.Pharm, Ph.D

Occurrence – Dried roots with 2-3 lateral

roots

Size – 20-30 cms and 6-12mm (diameter)

Shape – Straight, unbranched

Outer surface – Buff to grey yellow,

longitudinal wrinkles , at centre soft solid

mass with scattered pores

Odour – Characteristic

Taste – Bitter

As a root the following characters are seen

1. Cork – Cells are isodiametric and non

lignified

2.Starch – In cortex as well as in vascular

region, simple, oval in shape

3. Secondary xylem – Xylem fibres are

present

4. Xylem vessels – Reticulate thickened

5. Tracheids

Marker compound is Withaferin – A, which is

estimated by HPLC

SYNOPSIS :

Column – Poracil A coiled column (12feet X

1/8 th inch)

Mobile phase – n-hexane : isopropanol (9:1)

Flow rate – 0.2 ml/minute

Detector – 225nm (UV)

F.O.M – Not more than 2%

Total ash – Not more than 7%

Acid insoluble ash - Not more than 1.2%

Alcohol soluble extractive – Not less

than 20%

Alcohol (25%) soluble matter – Not

less than 16%

1.Standards specifications ;

Total ash: Not more than 4%

Loss on dryinig : Not more than 6%

Solubility : In water – Minimum 80%

and In 50% alcohol – 80%

Heavy metals : Not more than

20ppm

Aflatoxin : Negative

Microbial tests:

a. Total plate count - < 1000 cfu/gm

b. Yeast and moulds - < 100 cfu/gm

c. Coliform : Negative

d. Salmonella and Shigella - Negative

SYNOPSIS FOR TLC

Adsorbent – Silica gel G

Solvent system - For withanolides :

n.butanol: acetic acid: water(4:1:5)

For alkaloids: Toluene: ethyl acetate:

diethyl amine ( 70:20:10)

Detection:

Withanolides : Vanillin-sulphuric acid

reagent

Alkaloids : Drogendroffs reagent

Sample preparation:

Dissolve 0.25g of the extract in 25 ml of

methanol . Filter and concentrate the filtrate

to 5ml.

Identification:

Withanolides: 2 bluish violet spots with

identical Rf values in both extract and

standard withania root

Alkalods: 3 orange spots with identical Rf

values in both extract and standard withania

root

Alkaloids : A minimum of 0.75%

Withanolides : A minimum of 1.5%

Glyco-withanolides : A minimum of

2.5%

A. Macroscopic Stem – simple/ branched

Leaves – oblong with slightly asymmetric

base

Capsules – round , 1.8 cm diameter Seed – Longitudinal ribes ( 6 -7 ) and

transverse striations B. Microscopy Leaves- epidermis, stomata, calcium

oxalate, palisade cells

Stem – fibres, vessels

Fruit- epicarp shows anomocytic stomata

C. Identification:

Adsorbent – Silica gel GF 254

Solvent system – n-hexane: ethyl

acetate (2 :1)

Detection- sulphuric acid

Rf value –

Phyllanthin (0.28) and

Hypophyllanthin (0.37)

Column- C18- µ Bondapack

(30cmX3.9cm)

Mobile phase- Methanol: water (66:34)


Detector- 230mm ( UV )

Standard : Known concentration of

Phyllanthin and hypophyllanthin

( 0.05- 2 µg)


Total ash- Not more than 8%

Acid insoluble ash- Not more than

5%

Water soluble extractive- Not less

than 15%

n-hexane soluble extract- Not less

than 3%



Loss on dryinig : Not more than

6%

Solubility : In water – Minimum

80% and In 50% alcohol – 80%

Bulk density : 0.5-0.8

Microbial tests:



c. Salmonella and Shigella -

Negative

D.E.coli- Negative

Bitter as Lignan- Minimum of 2%

By HPLC- Phyllanthin and

hypophyllanthin

Estimation of total bitters as lignan by

gravimetry

A. Macroscopy Leaves –

Type – Simple and petiolated

Shape - orbicular - reniform

Margin- Crenate - denate

Apex- round with 5-7 nerved

Base – cordate

Flowers – small

Fruit – seeded and indehiscent

Odour – Charecteristic, Taste - Bitter

Mesophyll – Fragments are seen

Stomata – both Paracytic and Diacytic

Trichomes – absent

Calcium oxalate – rosette crytals

Mid rib – 3-5 vascular bundles, strips of

collenchyma

bellow the upper epidermis and above

lower epidermis


Solvent system

n-butanol: ethyl acetate: water (4:1:5 )

Detection:

Development of chromatogram - 12cm

Reference standard –

Rf value – cassoside (0.26), asiaticoside

(0.38) and brahmoside (0.58)

Column- C18- µ Bondapack (10cmX8mm)

Mobile phase- Acetonitrile: water (1:3)



Standard : Known concentration madecassoside (0.2-

4mg/ml) and asiaticoside in methanol ( 0.02- 0.4

mg/ml)

Sample preparation – 2 gm of drug powder reflux with

90% of methanol for 1hrs in boiling water bath. cool

and filter, collect the filtrate, concentrate the extract

and make up the volume to 50ml with methanol.

Procedure –

Known volume of both test and standards

are subjected to HPLC and the respective

peak areas for madecassoside and

asiaticoside are recorded.

Estimation –

Calculate their percentages by comparing

the recorded peak areas.



Acid insoluble ash- Not more than 7%

Saponin content – Not less than 1%



Loss on dryinig : Not more than 10%

Heavy metals – Not more than 20ppm

Microbial tests:



c. Salmonella and Shigella – Negative

A. Macroscopic :

Young stem – Green and smooth surface

Matured stem - Warty surface due to

lenticels

Fracture – Fibrous

Taste - Intensely bitter

Odour - Odourless

B.MICROSCOPIC :

Cork cells – Circular to isodiametric

Cortex cells – Collenchymatous

and parenchymatous

Xylem vessels – Cylindrical with

bordered pits

Tracheids - Bordered pits

Starch grains

C. Identification by TLC :


Solvent system – Chloroform : Methanol

(95:5)

Detection – Anisaldehyde – sulphuric acid

reagent and heating at 120 for 10 minutes

Test sample – Dissolve 0.25 g extract in

25ml pure methanol on a water bath ,

filter and concentrate to 5ml

Identification - Rf values 0.42 & 0.56

D. Estimation : By HPLC

Marker compound – Cordifolioside A

Column – C-18 Symmetry (30cmsX15mm)

Mobile phase – Gradient elution

Time – 0-15 mts – 95- 60% (Solvent A)

5- 40% (Solvent B)

- 15-30 mts- 60-0% (Solvent A)

40- 100% (Solvent B)

- 30-35 mts – 0% (Solvent A)

100% (Solvent B)

Flow rate – 1ml/minute

Reference solution – Cordifolioside A in

methanol ( 1mg in 2ml)

Test sample – Defat the powdered drug

in a soxhlet apparatus using petroleum

ether for 2hrs . Air dry the marc and

extract with methanol for 4hrs .Filter and

concentrate the filtrate to dryness under

vaccum and dissolve

the residue in 10ml of methanol.

Procedure :

Subject 10µl of reference solution and

test

sample to HPLC and record the

peaks.

Identify Cordifolioside A and calculate

the amount by comparing the

retention time and peak areas

respectively.

Quantitative standards :



Acid insoluble ash- Not more than 2%

Alcohol soluble extractive- Not less

than 3%

Water soluble extractive- Not less

than 11%

Total ash: Not more than 10% Loss on dryinig : Not more than 5% Solubility : In water – Minimum 80% and

In 50% alcohol – 70% Heavy metals – Not more than 20ppm Microbial tests: a. Total plate count - < 1000 cfu/gm b. Yeast and moulds - < 100 cfu/gm c. Salmonella and Shigella - Negative Coliform – Negative Pesticidal residue – Nil Aflatoxin - Nil

The amount of total bitters and

Cordifolioside A are estimated.

The amount of Cordifolioside A in

the dry extract determined by

HPLC method.

A. MACROSCOPY Type - Simple, opposite and

petiolate Size - 10-20 cm and 3-10cm Margin - Crenate Apex - Acuminate Base - Tapering Venation - Reticulate, 8-10 pairs of

lateral veins Petiole - 2-8cm Colour - Light green Odour - Charecteristic Taste - Bitter

Type – Dorsiventral leaf

Epidermis – Uniform cells with wavy

walls

Mesophyll – Distinct, palisade- two

layered

Stomata – Diacytic

Trichomes – Both covering and

glandular are present,

Covering trichomes – 2- 4 celled,

knee shaped

Glandular trichomes – sessile

( without stalk), quadricellur head.

Cystoliths – calcium carbonate

crystals

Mid rib – 3 - 5 vascular bundles, strips

of collenchyma bellow the upper

epidermis and above lower epidermis

SYNOPSIS FOR TLC


Solvent system

Toluene: Methanol : Dioxane : Ammonia

(1:1:2:5:0.5)

Detection:

Modified Dragendroffs reagent

Development of chromatogram - 12cm

Standard :

Known concentration in methanol

( 50- 80 µg/ml) .

Sample preparation – 1 gm of drug

powder reflux with methanol for 2hrs

, filter, collect the filtrate, concentrate

and acidified with dil Hcl. Follow the

general method of extraction of

alkaloids

Procedure –

Apply 5µl of reference and sample

Scanning – At 298nm and finger print

profiles can be recorded

Identification –

Saffron colored spot with a Rf value of

0.71

( vasicine) , both in sample and

standard.

Column- Resolvec C18- spherical

(3.9mmX 15cm)

Mobile phase- Methanol: water

(2:3)



Reference standard –

vasicine in methanol (1mg/ml)

Test sample –

1gm of drug is refluxed with

10ml of methanol, filter and

collect the filtrate , use for

sampling.

E.STANDARDS FOR CRUDE DRUG


Total ash - Not more than 17%

Acid insoluble ash - Not more than 1.0%

Water soluble extractive - Not less than

27%

Alcohol soluble extractive – Not more than

6%

Stomatal index – 10.8 – 18.2

Palisade ratio – 5.0- 8.5

Vasicine content – 0.4% by HPLC

basics of standerdisation by dr.u.srinivasa, professor and head, srinivas college of pharmacy,...

Education