The document discusses standardization of herbal drugs. It defines standardization as adjusting herbal drug preparations to a defined content of constituents with known therapeutic activity. Quality control is based on identity, purity, and content or assay. Various analytical methods like TLC, HPLC, and GC can be used to establish the constant composition of herbal preparations. The document also provides specifics on standardization of several individual herbal drugs.

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

2. 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.

3.  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?

4.  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

5.  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.

6.  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.

7.  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.

8.  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.

9.  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

10.  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.

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

12.  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

13.  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

14.  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)

15.  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%

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

17.  Microbial tests:
 a. Total plate count - < 1000 cfu/gm
 b. Yeast and moulds - < 100 cfu/gm
 c. Coliform : Negative
 d. Salmonella and Shigella - Negative

18.  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

19.  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

20.  Alkaloids : A minimum of 0.75%
 Withanolides : A minimum of 1.5%
 Glyco-withanolides : A minimum of 2.5%

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

22.  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

23.  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)

24. Column- C18- µ Bondapack (30cmX3.9cm)
Mobile phase- Methanol: water (66:34)
Flow rate – 1.8 ml/minute
Detector- 230mm ( UV )
Standard : Known concentration of
Phyllanthin and hypophyllanthin ( 0.05- 2
µg)

25.  F.O.M – Not more than 2%
 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%

26.  1.Standards specifications ;
 Total ash: Not more than 15%
 Loss on dryinig : Not more than 6%
 Solubility : In water – Minimum 80% and
In 50% alcohol – 80%
 Bulk density : 0.5-0.8

27.  Microbial tests:
 a. Total plate count - < 1000 cfu/gm
 b. Yeast and moulds - < 100 cfu/gm
 c. Salmonella and Shigella - Negative
 D.E.coli- Negative
 Bitter as Lignan- Minimum of 2%

28.  By HPLC- Phyllanthin and hypophyllanthin
 Estimation of total bitters as lignan by
gravimetry

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

30. 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

31.  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

32.  Adsorbent – Silica gel G
 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)

33.  Column- C18- µ Bondapack (10cmX8mm)
 Mobile phase- Acetonitrile: water (1:3)
 Flow rate – 1.5 ml/minute
 Detector- 205mm ( UV )
 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.

34.  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.

35.  F.O.M – Not more than 2%
 Total ash- Not more than 26%
 Acid insoluble ash- Not more than 7%
 Saponin content – Not less than 1%

36.  1.Standards specifications ;
 Total ash: Not more than 15%
 Loss on dryinig : Not more than 10%
 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

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

38. A. Macroscopic :
 Young stem – Green and smooth surface
 Matured stem - Warty surface due to
lenticels
 Fracture – Fibrous
 Taste - Intensely bitter
 Odour - Odourless

39. B.MICROSCOPIC :
 Cork cells – Circular to isodiametric
 Cortex cells – Collenchymatous and
parenchymatous
 Xylem vessels – Cylindrical with bordered
pits
 Tracheids - Bordered pits
 Starch grains

40. C. Identification by TLC :
 Adsorbent – Silica gel G
 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

41. 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)

42.  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.

43.  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.

44.  Quantitative standards :
 F.O.M – Not more than 2%
 Total ash- Not more than 11%
 Acid insoluble ash- Not more than 2%
 Alcohol soluble extractive- Not less than
3%
 Water soluble extractive- Not less than
11%

45.  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

46.  The amount of total bitters and
Cordifolioside A are estimated.
 The amount of Cordifolioside A in the dry
extract determined by HPLC method.

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

48.  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

49.  Type – Dorsiventral leaf
 Epidermis – Uniform cells with wavy walls
 Mesophyll – Distinct, palisade- two
layered
 Stomata – Diacytic
 Trichomes – Both covering and
glandular are present,

50.  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

51.  SYNOPSIS FOR TLC
 Adsorbent – Silica gel G
 Solvent system
 Toluene: Methanol : Dioxane : Ammonia
(1:1:2:5:0.5)
 Detection:
Modified Dragendroffs reagent
Development of chromatogram - 12cm

52. 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

53.  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.

54. Column- Resolvec C18- spherical
(3.9mmX 15cm)
Mobile phase- Methanol: water (2:3)
Flow rate – 0.7 ml/minute
Detector- 298mm ( UV )

55. 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.

56.  E.STANDARDS FOR CRUDE DRUG
 F.O.M – Not more than 2%
 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