AZVAGÀNDHAIIYDRO-ALCOHOLIC EXTRACT

A[vagandh lHydro-aleoholic Extract is a dried and powdercd 3ct preparcd frorm

per cent of total withane when assayed.
A[vagandh. The extract contains not less than l

Method of preparation:
extractor. Ad
Take A[vagandh suitably sizcd (powder or pieces) in an per cent aqteoUs
hefua
alcohol, about 3 tinmes the quantity of raw materialand heat under reflux at a terperature
S0-8s" for 3-4 hours. Filter the extract through a filter (preferably 10 un size) to a suitable
into the carse
sized vessel. The marc is extracted three times more, filtering the extract eact ime
vessel. Concentrate the combincd filtrate to åsyrupy consistency and dry under vacuum (betwern
below 5 per cent M:l1
400-600 mm of Hg) at atemperature not excecding 80 till the moisture is
the mass and sieve the powder through 500 um mesh to obtain the extract and pack. The vicla
obtained is about 16 per cent.

ldentity, Purityand Strength:

Thin-layer chromatography:
Carry out thin-layer chromatography 254 nm Visible after derivatisation
on aprecoated silica gel 60Fs, plate
(Appendix 3.5) using withaferin A as a R
reference standard. Solvent system: 1.0
Chloroforn : methanol (9.0 : L.0).
Test solution: To 3 g of the substance
being examined, add 25 ml of
methanol. heat on a water bath for
10-15 minutes, cool and filter.
0.5
Standard solution: Dissolve 10 mg of
witlhaferin A in l0 ml of methanol.
Procedure: Apply 10 l each of the
test and standard solutions on a TLC
plate as bands of 10 mm. Develop the
plate to a distance of 8 cm from the 0.0
line of application. Dry the plate in air
and examine undcr 254 nn. Spray the RS RS
plate with a solution of anisaldehyde Fig.1. Thin-Layer Chromatogrann of
sulphuric acid reagent. Heat the plate A[vagandh+hydro-alcobolie extract
ut 110" or about 5 minutes or till the RS: Withaferin A, T: Test solution
bands are clearly visible. The chromatogram obtained with test
solution shows a band at
corresponding to that of withaterin A and the profile should be similar to the one given in R0.30
the TLC
(Fig. l).

34
 Ari
Quantitative parameters:
Loss ondying: Not inore than 5.0 per
Total ash: cent, Appendix 2.14
Not more than 16.0 per
Acid-insoluble ash: cent, Appendix 2.1.5
Not more than 2.0 per cent, Appendix 2.1.7
plt: 4.5 - 5.5,
Total soluble solids: Appendix 2.1.10
Not less than 90.0per cent, Appendix 2.1.11
(Method-)
Other requirements:
Heavy metals:
Complies with the prescribedlimits, Appendix 3.1
Microbial contamination: Complies with the prescribed limits, Appendix 3.2
Pesticide residues:
Complies with the prescribed limits, Appendix 3.3
Residual solvent: Complies with the pre_cribed limits, Appendix 3.8
Aflatoxins:
Complies with the prescribed limits, Appendix 3,4
Assay:
Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g,
accurately weighed, of the substance being examined. Add 50ml of nethanol, reflux on a water
batl1 for I5 minutes, cool and filter. Reflux the residue further with 50 ml of mnethanol for two
times more, cool and filter. Combine the fltrates, concentrate, and make up the volume in a
100-nl volumetric Ilask. Standard solution: Take about 10 mg, accurately weighed, each of
withanoside IV RS and withanolide A RS in a 100-ml volumetric flask and dissolve in 50 ml of
metha:nol and make up the volume with methanol, Chromatographic system: High performance
liquid chromatugraphy. Column and stationary phase; C18 (250 mm x 4.6 mm), 5 um. Mobile
phase: Filtered and degassed gradient misture of phosphate buffer (prepared by dissolving 0.14 g
of potassiunn dihydrogen orthophosphate in 500 ml of water, adding 0.5 ml of orthophosphoric
acid and making up the volume to 1000 ml) and acetonitrile in the following proportions:
Time Phosphate buffer Acetonitrile
(minutes) (per cent) (per cent)
0.01 95
n8?nn 8
18 55 45

25 20 80
28 20 80
55 45
35
40 95
45
95

Procedure: Iniee
lnjection volume: 20 ul. Flow rate: 1.5 ml per minute. Detection: UV, 227 nn.
20 Lul of the filered standard solution and record the chromatogram. Inject 20 ul of the filtered
retention
icst solution, record the chronatogranm and identify lhc analyte peaks using the relative
limes, as bclo

35
 Relative retention time
Analyte
Withanolideglycosides 0.70
Wihanosidc IV 0.89
Withanoside V& VI
Withanolide aglycones 0.96
12-Deoxywithastramonolide
1.00
Withanolide A
1.14
WithanolideB
IV by ssunming thc
withanolide glycosidcs in the sample as withanoside IV in
Calculatethe content of from the declarcd content of wilhanoside
Vand VI and
l areas of withanoside IV,
of withanolide aglyconcs in the sample as withanolide A
ih2noside IV RS.Calculate the content
deoxywithastramonolide, withanolide A and withanolide B and
of12- withanolida
by sununing the peak arcas withanolide A in withanolide A RS. Sum the content of
fronm the declared content of
to get total withanolides.
1nDeovvwithastramonobde
ciycosides and withanolide aglycones
Wihanolide-A
ywitha
Withanoside
V6
withafcrin
Standard mix

Withanoside IV and Withanolide A RS

ASU A[vagandhã hydro-alcoholic extnt

IV
Withanoside -Withanolide-B

10

extract with
Fig.2. HPLC chromatograms of A[vagandh hydro-alcoholic
Withanoside IV, WWithanolide A as RS and Standard mix

Additional requirements:
light, moisture nd
Packaging and Storage: Preserve in well closed containers, protected from
store at room tempcraturc.
Labeling: The label states the official name, following the Latin binomial, the part of the pla
uscd and typeof extract.
APl reference standards: API Witlhaferin ARS, Withanoside IV RS and Withanolide ARS

36