Compilation of few herbal drugs and formulation

1. ANALYSIS OF HERBAL
DRUGS BY TITRIMETRY
Prepared by-
Vedshree Raole
Guided by-
Dr.S.J Rajput
Faculty of Pharmacy,
M. S. University

2. CONTENTS
 Definitions
 Introduction to Titrimetry
 Estimation of Marketed Extracts.
 Estimation of Marketed tablets.
 Estimation of Excipients in herbal
formulation
 Estimation of Other Marketed
Formulations.
 References

3. Definitions
 Crude herbs: This term means, unless specified
otherwise, mainly whole, fragmented or cut, plants,
parts of plants, algae, fungi, and lichen in a form which
is not processed.
 Processed herb: Processed herbs means preparations
obtained by subjecting herbs to treatment such as
extraction, distillation, expression, fractionation,
purification, concentration and partial or full
fermentation.
 Dry Extract: Dry extracts usually have a loss on drying
or water content not greater than 5 per cent w/w,
unless specified otherwise in any monograph.
 Tincture: would normally mean an extract where
aqueous-ethanol is used as a solvent for extraction.

4. Standardization as perWHO: Measures
taken during the manufacturing and
Quality control leading to the
reproducible quality.
 It can be achieved by adjusting the extract
with approved inert materials or blending
one or more batches of extracts.

5. TITRIMETRY
 Titration is a method for determining the concentration of a solution by
reacting a known volume of that solution with a solution of known
concentration.
 A neutralization reaction is a reaction in which an equal molar quantities of
acid and a base in an aqueous solution react to produce a salt and water.
 Complexometric titration (chelatometry) is a form of volumetric analysis in
which the formation of a colored complex is used to indicate the end point of
a titration.
 Non- aqueous titrations are the titrations in which weakly acidic or basic
substances are carried out using non–aqueous solvents to get sharp end
point.The moisture content in non–aqueous titrations should not be more
than 0.05%.
 Redox reactions are the oxidation-reduction reactions in which a
change in the valency of reacting elements takes place.Titrations in
which titrant and analyte undergo redox reactions.

6. Estimation of Tannins in
Amla Juice powder[1]
 Weigh accurately about 2 g of the extract, add 50 ml boiling water and heat
it on a water-bath for 30 minutes with frequent stirring. Allow the solution
to settle and carefully decant it through a piece of cotton wool to a 500-ml
volumetric flask.
 Repeat the extraction for 5 times with 50 ml of boiling water. To confirm
that all tannins have been extracted, add 3-4 drops of ferric ammonium
sulphate solution to 5 ml of the extract. Blue colour will not be produced, if
all tannins have been extracted. If blue colour develops extract again with 2
times with 50 ml of boiling water and check with ferric ammonium sulphate
solution again. Cool the extracts and make up to the mark with water.
 Take 50 ml into a 250-ml conical flask and add 50 ml of indigo sulphonic
acid solution(prepared by dissolving 1 g of indigo carmin in 50 ml of
sulphuric acid, add 500 ml of water and dilute this solution to 1000 ml.)
Titrate, with 0.1 M KMnO4 using indigo sulphonic acid as indicator until a
golden yellow colour is produced. Carry out a blank titration.
 1 ml of 0.1 M KMnO4 is equivalent to 0.004157 g of polyphenoIs calculated
as tannic acid.

8. Estimation of dried
Belladona Leaf tincture[1]
 Evaporate 50.0 g of the tincture under examination to a volume of
about 10 ml. Transfer quantitatively to a separating funnel, with the
minimum volume of ethanol (70 per cent v/v). Add 5 ml of strong
ammonia solution and 15 ml of water.
 Extract with three quantities, each of 40 ml of a mixture of 1 volume of
dichloromethane and 3 volumes of peroxide-free ether, carefully to
avoid emulsion, if the alkaloids are completely extracted. Combine the
dichloromethane and ether extracts; concentrate the solution to a
volume of about 50 ml by heating on a water-bath. Transfer the
resulting solution quantitatively to a separating funnel, rinsing with
peroxide-free ether.
 Add a quantity of peroxide-free ether equal to at least 2.1 times the
volume of the solution to produce a layer having a density well below
that of water. Extract the resulting solution with minimum of three
quantities, each of 20 ml of 0.25 M sulphuric acid until the alkaloids are
completely extracted.
 Separate the layers by centrifugation if necessary and transfer the layers
to a separating funnel. Make the combined layers alkaline with strong
ammonia solution and extract with minimum of three quantities, each of
30 ml of dichloromethane until the alkaloids are completely extracted.

9.  Combine the dichloromethane and ether extracts, add 4 g
of anhydrous sodium sulphate and allow standing for 30
minutes with occasional shaking. Decant the
dichloromethane and filter. Wash the sodium sulphate
with three quantities, each of 10 ml of dichloromethane.
Combine the dichloromethane and ether extracts;
evaporate to dryness on a water-bath. Heat the residue
in an oven at 105° for 15 minutes.
 Dissolve the residue in a few ml of dichloromethane,
evaporate to dryness on a water-bath and heat the
residue in an oven at 105° for 15 minutes again. Dissolve
the residue in a few ml of dichloromethane. Add 20.0 ml
of 0.01 M sulphuric acid and remove the
dichloromethane by evaporation on a waterbath.
 Titrate the excess of acid with 0.02 M sodium hydroxide
using methyl red mixed solution as indicator.
 1 ml of 0.01 M sulphuric acid is equivalent to 0.005788 g
of total alkaloids calculated as hyoscyamine.
 Calculate the content of total alkaloids with reference to
the dried material.

11. Estimation of Calcium in
Garcinia Aq. Extract[1]
Calcium -
 Test solution. Weigh accurately about 0.2 g of
the extract and transfer into a 500 ml conical
flask. Dissolve in 2 ml of 3 M hydrochloric
acid and add 200 ml of water. Add 15 ml of 1
M Sodium hydroxide and titrate with 0.05 M
EDTA using hydroxy naphthol blue as
indicator until deep blue colour persist.
 Each ml of 0.05MEDTA solution is equivalent
to 0.002004 g of calcium.

13. Estimation of Papain in
Carica Papaya extract[1]
 Weigh accurately about 0.5 g, triturate with 10 ml of
cysteine hydrochloride solution and dilute to 100.0 ml
with water.
 To 30 ml of water in each of two flasks add 15.0 ml of
casein solution and maintain at 60° by heating on a
water-bath.
 To the first flask add 5 ml of solution under
examination and to the second flask- add 5.0 ml of
the same solution, previously boiled for 2 minutes and
cooled. Maintain the solutions at 60° for 30 minutes,
cool rapidly to room temperature.
 Add to each flask 0.75 ml of phenolphthalein solution
and 10 ml of formaldehyde solution, previously
neutralised to phenolphthalein solution.
 Titrate both solutions with 0.1 M sodium hydroxide
to the same definite pink colour; the difference
between the two titrations is not less than 4.5 ml.

15. Estimation of Peppermint
Oil[1]
For esters –
 To 2 g in a borosilicate glass flask add 2 ml of ethanol (90
per cent) and 0.25 ml of phenolphthalein solution,
neutralise with 0.5M ethanolic potassium hydroxide.
 Add an additional 25.0 ml and a little pumice powder or
a few pieces of porous pot and heat under a reflux
condenser on a water-bath for 30 minutes.
 Add 1 ml of phenolphthalein solution and immediately
titrate with 0.5M hydrochloric acid.
 Repeat the operation without the substance under
examination. The difference between the titrations
represents the volume of alkali required to saponify the
esters.
 1 ml of 0.5 M ethanolic potassium hydroxide is equivalent
to 0.09915 g of esters, calculated as menthyl acetate,
CI2H2202.

16. For free alcohols –
 To 1 g in a dry, 150-ml acetylation add 3 ml of a mixture of 3
volumes of pyridine and 1 volume of acetic anhydride. Determine
the weight of the acetylation mixture to the nearest mg, keeping the
flask closed while weighing.
 Boil under a reflux condenser in a water-bath for 3 hours,
maintaining the water level 2 to 3 cm above the level of the liquid in
the flask throughout. Remove the flask from the water-bath and add
50 ml of water through the condenser. Remove the condenser and
wash the walls of the flask with 10 ml of water.
 Allow to stand for 15 minutes and titrate with 0.5 M sodium
hydroxide using 1 ml of phenolphthalein solution as indicator. Repeat
the operation without the substance under examination.
 The difference between the titrations represents the volume of
sodium hydroxide required.
 1 ml of 0.5 M sodium hydroxide is equivalent to 0.07815 g of free
alcohols, calculated as menthol, CIOH2oO.
 If the quantities of acetic anhydride in pyridine used in the two
determinations differ by more than 5 mg, adjust the volume of alkali
used in the second titration by multiplying with a/b where a is the
weight, in g, of acetic anhydride in pyridine used in the first
determination and b is the weight, in g, of acetic anhydride in
pyridine used in the second test.

17. For ketones-
•To 2 g add 25 ml of a 5.0 %w/v solution of hydroxylamine
hydrochloride in ethanol (95 per cent), heat on a water-bath for 1 hour,
allow to cool, add about 1mg of methyl orange.
•Titrate with 0.5 M ethanolic KOH until an orange-yellow colour is
obtained.
•Repeat the heating for further periods of 1 hour until, after cooling, not
more than 0.1 ml of 0.5 M ethanolic potassium hydroxide is required to
neutralise the solution.
•1 ml of 0.5 M ethanolic potassium hydroxide is equivalent to 0.07710 g
of ketones, calculated as menthone, CIOH1SO.

18. Estimation of Acid value
in Shellac[1]
Acid value (2.3.23). 50 to 70, determined by the following method.
• Weigh accurately about 2.0 g and dissolve with the aid of gentle heat, in 50 ml
of ethanol (95per cent) previously neutralised to ethanolic thymol blue solution.
•Titrate with 0.1 M ethanolic potassium hydroxide using ethanolic thymol blue
solution as an external indicator.
• Calculate the acid value from the expression
5.61 x a/w
where, a number of ml of 0.1 M ethanolic potassium
hydroxide and
w = weight, in g, of the sample.

20. Estimation of Ephedrine in
marketed formulation[2]
 Weigh 20 tablets and reduce to a fine powder.
Weigh accurately a quantity of the powder
equivalent to about 0.15 g of Ephedrine
Hydrochloride and add 30 ml of anhydrous
GAA and 10 ml of mercuric acetate solution.
Warm gently to effect solution, cool and for
non-aqueous titration, using 0.1 ml of crystal
violet solution as indicator, until the violet
colour changes to green-blue.
 Perform a blank determination and make any
necessary correction.
 Each ml of 0.1M perchloric acid is equivalent to
0.02017 g of C10H15NO,HCl.

22. Estimation of cinnamic acid
and benzoic acid in tolu
balsam marketed preperation[2]
 Weigh accurately about 1.25 g and boil with 25 ml of dilute
ethanolic potassium hydroxide solution under a reflux condenser for
1 hour. Remove the ethanol and digest the residue with 50 ml of hot
water until diffused. Cool the liquid, add 150 ml of water and 1.5 g
of magnesium sulphate dissolved in 50 ml of water.
 Mix thoroughly and set aside for 10 minutes. Filter, wash the residue
on the filter with 20 ml of water, acidify the combined filtrate and
washings with HCl and extract with successive quantities of 50, 40,
30, 30 and 30 ml of ether.
 Combine the ether extracts and discard the aqueous portion. Extract
with successive quantities of 20, 20, 10, 10 and 10 ml of sodium
bicarbonate solution, washing each aqueous extract with the same
20 ml of ether. Discard the ether layers, acidify the combined
aqueous extracts with hydrochloric acid and extract with successive
quantities of 30, 20, 20 and 10 ml of chloroform, filtering each
chloroform extract through a plug of cotton wool on which a layer
of anhydrous sodium sulphate is placed.

23.  Evaporate the chloroform on a water-bath until
about 10 ml remains and remove the remainder in a
current of air stopping immediately when the last
trace of solvent is removed.
 Dissolve the residue by warming with 10 ml of
ethanol (95%), previously neutralised to phenol red
solution, cool and titrate with 0.1M sodium
hydroxide using phenol red solution as indicator.
 Each ml of 0.1M sodium hydroxide is equivalent to
0.01482 g of total balsamic acids,calculated as
cinnamic acid, C9H8O2, in Sumatra Benzoin and
0.01221 g of total balsamic acids, calculated as
benzoic acid, C7H6O2, in Siam Benzoin.

25. Estimation Alkaloid
content in Kutuja [2,4]
 Kutaja contains NLT 2% of total alkaloids when assayed by the
following method:
 Weigh accurately about 5 g in powder (No. 85 sieve) and moisten with
10 ml of an Alcohol-chloroform mixture (1 :3) containing 2% of
Ammonia solution for 15 minutes.
 Pack the mixture in a small glass percolator surrounded by a jacket of
hot water kept at 50°. Macerate with more of the alkaline Alcohol-
chloroform mixture for an hour and collect 25 ml of percolate in a
receiver containing 1 g of Oxalic acid dissolved in 5 ml of alcohol.
 Stop the percolation add l0 ml of the alcohol chloroform mixture
containing 1% w/v of NaOH and macerate for fifteen minutes. Continue
the percolation adding further quantities of the alcohol chloroform
mixture until the alkaloids are completely extracted. Mix the percolate
well and extract by shaking with five 20 ml portions of 2 N
Hydrochloric acid. Combine the acid extracts and make alkaline with
dilute Ammonia Solution.

26.  Extract with four 10 ml portions of Chloroform, add
1 ml of 0.5 N Sodium Hydroxide, and extract again
with Chloroform. Wash each Chloroform extract
with the same two 10 ml portions of water
contained in different separators.
 Combine the Chloroform extracts, add 20 ml of
O.IN Sulphuric Acid and shake well for 5 Minutes.
Transfer the acid Liquid to a conical flask, wash the
Chloroform extract with two 20 ml portions of
water and add the washing to the acid liquid in the
conical flask.
 Titrate the excess of acid with 0.1N Sodium
Hydroxide using the mixed 3 indicator.
 Each ml of 0.1N Sulphuric Acid is equivalent to
0.01657g of total alkaloids of Kutaja.

28. Estimation of Strychnin
in Strychnous Nux
Vomica [2]
 Weigh accurately about 10g in fine powder, add 100 ml of
a 33% v/v mixture of chloroform in solvent ether and set
aside for ten minutes.
 Add 5 ml of dilute ammonia solution and shake
continuously for six hours. Transfer to a continuous
extraction apparatus with more of the same solvent
mixture and extract for two hours.
 Filter the solvent extract, washing the filter with solvent
ether and extract with successive quantities of 20 ml, 20
ml,10 ml and 10 ml of 1N sulphuric acid, until complete
extraction of the alkaloids is affected. Combine the acid
extracts and make alkaline with dilute ammonia solution.
 Extract with successive quantities of 20 ml, 20 ml and 10 ml
of chloroform until complete extraction of the alkaloids is
effected. Evaporate the chloroform, add 5 ml of alcohol
and evaporate to dryness.

29.  Dissolve the residue in a mixture of 15 ml of a 3% w/v solution of
sulphuric acid and 2 ml of nitric acid, add a few crystals of sodium nitrite
and set aside at 18°C for thirty minutes.
 Transfer to a separator containing 20 ml of solution of NaOH, shake for
two minutes and then shake with 20 ml of chloroform, separate the
chloroform solution, wash it with 5 ml of solution of NaOH and then
with two quantities each of 10 ml of water.
 Continue the extraction with successive quantities of 10 ml of
chloroform, until complete extraction of the alkaloids is effected,
washing each chloroform solution separately with the 5 ml of solution of
sodium hydroxide and with the two quantities of water, which were
used for washing the first chloroform solution.
 Titrate the second wash with 0.1 N sulphuric acid using solution of
methyl orange as indicator if more than 0.1 ml is required, wash the
combined chloroform solutions with further quantities, each of 10 ml of
water until on titration not more than 0.1 ml of 0.1 N sulphuric acid is
required.
 Remove the chloroform, add 5 ml of alcohol, evaporate, and dry for
thirty minutes, at 100°C. Dissolve the residue in 10 ml of 0.1 N sulphuric
acid and titrate the excess of acid with 0.1 N sodium hydroxide, using
solution of methyl orange as indicator.
Each ml of 0.1 N sulphuric acid is equivalent to 0.03344 g of strychinine,
multiply the result by 1.02 to correct for loss of strychinine.

31. Presence of sodium benzoate in
commercially available samples
of Dasamoolarishta[13]
 Benzoic acid was separated from a known quantity of sample
by saturating with NaCl, and acidifying with dilute HCl.
 This sample was extracted with chloroform. The chloroform
layer was made mineral acid free and the solvent was
removed by evaporation.
 The residue was dissolved in neutral alcohol and the amount
of benzoic acid determined by titration against standard alkali.
The amount of benzoic acid present in the sample was
determined using the formula
Benzoic acid (ppm) = 122 X titre X dilution X 1000 X NaoH
Weight of sample

33. STANDARDIZATION OF
PANCHATIKVA GHRITA [8]
Acid Value:
 10gm of sample is taken in a 250 ml volumetric flask and 50ml of
mixture of equal volumes of ethanol and solvent ether are added and
is neutralised after addition of 1 ml of phenolphthalein solution.
 This is heated gently on water bath until the substance is completely
melted.
 It is then titrated against 0.1N potassium hydroxide solution with
continuous shaking until pink color which is persistent for 15 seconds
is obtained.
 Number of ml is noted and calculation is done using the following
equation:
Acid value= a×0.00561 ×1000/W
where,
a= number of ml of 0.1N KOH required
W= Weight of sample in gm

34. Iodine Value:
 Wij’s method is used for determination of Iodine value.
 10ml of iodine monochloride is mixed with 1800 ml of GAA and shaken vigorously.
 2g of sample is taken in Erlenmayer flask, 25 ml of carbon tetrachloride is added and
the content is mixed well. To this 25ml of Wij’s solution is pipetted and the flask is
stoppered with glass stopper previously washed with KI solution.
 It is swirled for proper mixing and kept in dark for 1 hour. Blank is prepared in the
same manner.
 15ml of KI is added followed by 100 ml of freshly boiled and cooled water. Rinsing of
stopper is also done.
 The liberated iodine is titrated against standardised sodium thiosulphate solution using
starch indicator until persistent blue color is obtained.
 Iodine value is calculated as:
Iodine value= 12.69(B-S) N/W
where,
B = ml of sodium thiosulphate required by blank
S = ml of sodium thiosulphate required by sample
N = Normality of sodium thiosulphate
W = Weight of sample taken

36. Quantitative Estimation of
tannin (gallotannic acid) in
marketed ayurvedic churna
preparations [5]
 Pipette out 10ml from the stock solution and add 10ml indigo carmine
indicator solution and make up volume up to 100ml with distilled
water. Heat this solution on water bath for 15-20 min at 70oc and
titrated against 0.1N KMnO4 solution which has taken in burette. Note
the Colour changes from blue to parrot green and finally the
appearance of golden yellow color at end point. The consumed
volume of 0.1N KMnO4 is recorded as A. Take another 2 reading using
same procedure and take mean out of that. Blank reading taken by
replacing sample solution with distilled water and follow the same
procedure as above. Note down the ml of KMnO4 is used in blank as
B
%Tannins = (A - B)*250 *100*(0.00425 gm tannins/ml of 0.1N KMnO4)
gm of sample powder * ml of sample taken

38. Determination of ethanol
content of the Abhayarishta by
Redox titration [7]
 The method uses a redox titration to find the concentration of
ethanol in the aqueous solution.
 The ethanol gets oxidized to ethanoic acid by reacting it with excess
of potassium dichromate in concentrated sulphuric acid.
 Transfer 10 mL of the acid dichromate solution to a 250 mL conical
flask with matching rubber stopper.
 Pipette 1 mL of the diluted sample into the sample holder. This can
be a 5mL beaker or glass vial. Prepare three samples of the beverage
as the entire contents of the flask are used in the titration.
 Suspend the sample holder over the dichromate solution and hold in
place with the rubber stopper.
 Store the flask overnight at 25–30°C (an incubator is ideal).
 Next morning allow the flask to come to room temperature, then
loosen the stopper carefully and remove and discard the sample
holder.

39.  Rinse the walls of the flask with distilled water,
then add about 100 mL of distilled water and 1 mL
of potassium iodide solution. Swirl to mix.
 Prepare 3 blank titrations by adding 10 mL of acid
dichromate solution to a conical flask, adding 100
mL of water and 1 mL of potassium iodide solution
and swirling to mix.
 Fill a burette with sodium thiosulfate solution and
titrate each flask with sodium thiosulfate. When
the brown iodine colour fades to yellow , add 1
mL of starch solution and keep titrating until the
blue colour disappears Titrate the blank flasks first,
and repeat until concordant results.

41. Determination of reducing
sugars in
Marketed Kumari Asava [11]
 20ml of kumari asava was taken and neutralize with
NaOH. The neutralize solution was evaporated to
half volume on waterbath at 50˚C to removed
alcohol. After cooling 10 ml of 21.9 g zinc acetate,
3ml GAA followed by 10.6 g potassium ferrocyanide
and distilled water was added to make a volume of
100ml.
 10ml of Fehling solution was taken and burette
solution was added drop wise and heat to boiling
over hot plate till blue color appeared. At this time,
two drops of methylene blue was added and the
titration was carried on till brick red color was
obtained.

43. References
1. The Indian pharmacopoeia, IP ‘10, volume-3, Indian
pharmacopoeia commission, Ghaziabad, India, 1996. Pg No.:
744-830
2. Ayyurvedic pharmacopoeia Vol-I part-1 Pg no:119-120, part-4 Pg
No:170-171.
3. WHO guidelines for assessing quality of herbal medicines with
reference to contaminants and residues
4. Identification, evaluation and standardization of herbal drugs: A
review Archana Gautam*, Shiv Jee
Kashyap(http://scholarsresearchlibrary.com/archive.html)
5. Validated simple redox titration method for the estimation of
gallotannins in marketed ayurvedic churna preparations Ankit V.
Patel*, Kalpen N. Patel and Maulika S. Patel J. Chem. Pharm.
Res., 2011, 3(6):293-299

44. 6. Development of Evaluation Parameters for Balchaturbhadra Churna &
Comparision with Market Formulation Shahebaz N. Ghadiyali*, Nitesh
Patel, Nikunj Trivedi , Hetal Desai, IJPRAS Volume 1, issue 4 (2012),75-84
7. STANDARDIZATION OF ABHYARISHTA AS PER WHO GUIDELINES
Shreyansh S. Mutha, Veena S. Kasture*, Seema A. Gosavi, Rasika D
Bhalke.,Sarita S. Pawar WJPPS Volume 3, Issue 1, 510-518.
8. Pharmacoanalytical study and Standardization of Panchatikta Gritha,
Haldar Pranob, IRJP 2013,4(9)
9. http://www.outreach.canterbury.ac.nz/chemistry/ethanol.shtml
10. Pharmaceutical Drug Analysis by Ashutosh Kar
11. STANDARDIZATION OF MARKETED KUMARI ASAVA- A POLYHERBAL
AYURVEDIC FORMULATION DS. Chumbhale1*, SR. Chaudhari1 and CD.
Upasani IJPCBS 2014, 4(3), 681-685
12. DETERMINATION OF TANNINS CONTENT BY TITRIMETRIC METHOD
FOR COMPARISON OF DIFFERENT PLANT SPECIES M. Atanassova, V.
Christova-Bagdassarian
13. A study on the presence of sodium benzoate in commercially available
samples of Dasamoolarishta- an ayurvedic preparation Nishna KP, Robin
PC, Harikumar R and Jayachandran VP*, IJPCS, ISSN: 2277-5005.

46. Estimation of Acidity in Starch
 Add 10.0 g to 100 ml of ethanol (70 per cent)
previously neutralised to phenolphthalein
solution, shake for 1 hour, filter.
 Titrate 50 ml of the filtrate with 0.1 M sodium
hydroxide.
 Not more than 2.0 ml is required to change the
colour of the solution.