2. USP
It is published by the United State Pharmacopoeial
convention, Inc., Rockville, Maryland, USA.
It provides standards for drugs and chemicals used in
the practice of medicine and pharmacy.
3. 1. Acacia
Acacia is the dried gummy exudate from the stems and
branches of Acacia senegal (Linné) Willdenow or of
other related African species of Acacia (Fam.
Leguminosae).
Packaging and storage— Preserve in tight containers.
Solubility and reaction— Dissolve 1 g in 2 mL of
water; the resulting solution flows readily and is acid
to litmus.
4. Conti…..
Botanic characteristics—
Acacia— Spheroidal tears up to 32 mm in diameter or
in angular fragments of white to yellowish white color.
Is translucent or somewhat opaque from the presence of
numerous minute fissures; very brittle, the fractured
surface glassy and occasionally iridescent.
Is almost odorless and produces a mucilaginous
sensation on the tongue.
5. Conti…
Flake Acacia— White to yellowish white, thin flakes,
appearing under the microscope as colorless, striated
fragments.
Powdered Acacia— White to yellowish white, angular
microscopic fragments with only traces of starch or
vegetable tissues present
Granular Acacia— White to pale yellowish white, fine
granules. Under the microscope it appears as colorless,
glassy, irregularly angular fragments up to 100 µm in
thickness, some of which exhibit parallel linear streaks.
Spray-dried Acacia— White to off-white compacted
microscopic fragments or whole spheres.
6. Conti….
Identification— To 10 mL of a cold solution (1 in 50)
add 0.2 mL of diluted lead subacetate TS: a
flocculent, or curdy, white precipitate is formed
immediately.
Microbial limits — It meets the requirements of the
test for absence of Salmonella species.
Water, Method III — Dry it at 105⁰C for 5 hours: it
loses not more than 15.0% of its weight. For
unground Acacia, crush it in a mortar until it passes
through a No. 40 sieve, and mix the ground material
before weighing the test specimen.
Total ash : not more than 4.0%.
Acid-insoluble ash: not more than 0.5%.
7. Conti….
Insoluble residue— Dissolve 5.0 g of powdered or
finely ground Acacia in about 100 mL of water in a
250-mL conical flask, add 10 mL of 3 N hydrochloric
acid, and boil gently for 15 minutes. Filter by suction,
while hot, through a tared filtering crucible, wash
thoroughly with hot water, dry at 105 for 1 hour, and
weigh. The weight of the residue thus obtained does
not exceed 50 mg
Arsenic, Method II : 3 ppm.
Lead : 0.001%
8. Conti….
Heavy metals, Method II : 0.004%.
Starch or dextrin— Boil a solution (1 in 50), cool, and
add iodine TS: no bluish or reddish color is produced.
Organic volatile impurities, Method I: meets the
requirements
Tannin-bearing gums— To 10 mL of a solution (1 in
50) add 0.1 mL of ferric chloride TS: no blackish
coloration or blackish precipitate is produced.
9. 2. Ashwagandha
DEFINITION: Ashwagandha Root is the dried mature
roots of Withania somnifera (L.) Dunal (Fam.
Solanaceae).
It contains NLT 0.3% of withanolides, calculated on the
dried basis as the sum of withanolide aglycones,
calculated as withanolide A, and withanolide
glycosides, calculated as withanoside IV.
10. Conti..
IDENTIFICATION
A. Thin-Layer Chromatographic Identification Test
Standard solution: About 200 mg of USP Powdered
Ashwagandha Root Extract RS in 10 mL of methanol. Heat
gently for 10–15 min, centrifuge, and use the supernatant.
Sample solution: Transfer about 5.0 g of Ashwagandha
Root, finely powdered, to a 250-mL flask fitted with a
reflux condenser. Add 50 mL of methanol, reflux on a water
bath for 10–15 min, cool to room temperature, and decant
the supernatant. Repeat until the last extract is colorless.
Combine the extracts, filter, concentrate under vacuum to
about 40 mL, and adjust the volume with methanol to 50.0
mL.
11. Conti…
Adsorbent: 0.25-mm layer of chromatographic silica gel
Application volume: 25 µL
Developing solvent system: A mixture of ethyl acetate, toluene,
and acetic acid (45:55:3)Spray reagent: Mix 0.5 mL of
anisaldehyde, 10 mL of glacial acetic acid, 85 mL of methanol,
and 5 mL of concentrated sulfuric acid in the order given.
Analysis Samples:
Standard solution and Sample solutionApply the Samples as
bands to a suitable plate (see Chromatography 621). Use a
saturated chamber. Develop until the solvent front has moved up
about 90% of the length of the plate. Dry the plate, spray with
Spray reagent, heat for 5–10 min at 100⁰C and examine under
visible light.
12. Acceptance criteria:
The Sample solution exhibits five main grayish-blue
bands with RF values of approximately 0.12, 0.29,
0.47, 0.67, and 0.73 that are similar in position and
color to the main bands from the Standard solution.
Other less intense bands are observed for the Sample
solution and the Standard solution.
13. B. The Sample solution in the test for Content of
Withanolides shows main peaks at retention times
corresponding to those of withanolide A and
withanoside IV in Standard solution A and Standard
solution B, respectively.
Identify other withanolide peaks in the Sample solution
by comparison with Standard solution C and the
reference chromatogram provided with the lot of USP
Powdered Ashwagandha Root Extract RS being used.
The Sample solution shows additional peaks
corresponding to some of the following withanolides:
physagulin D, 27-hydroxywithanone, withanoside V,
withanoside VI, withaferin A, withastramonolide,
withanone, and withanolide B.
14. Conti..
Histology
Transverse section of roots: It shows a narrow band of yellowish
crumpled cork, moderate-size cortex and a wide wood.
The cork cells are rectangular, radially flattened, nonlignified, and
filled with starch grains and reddish brown content; cork cambium is
2–4 diffused rows of cells; secondary cortex is formed of 20–25 rows
of thin-wall parenchymatous cells, filled with starch grains, and
shows occasional microsphenoidal crystals of calcium oxalate;
phloem consists of sieve tubes, companion cells, and phloem
parenchyma; vascular cambium consists of tangentially elongated
parenchymatous cells; vessels and tracheids are in radial rows toward
the periphery of the wood; medullary rays are uniseriate to 2- to 3-
seriate, and are filled with starch grains; scattered vessels in groups
are embedded in the parenchyma; vessels have pitted and scalariform
thickening, and generally the end walls are perforated; and a few
fibers with thick lignified walls are also found scattered in the wood.
15. Conti..
Loss on Drying : Dry 1.0 g of finely powdered
Ashwagandha Root at 105 for 3 h: it loses NMT 12.0%
of its Origin, Total Ash 561: NMT 7.0%, determined
on 1.0 g of finely powdered Ashwagandha Root
Articles of Botanical Origin, Alcohol-Soluble
Extractives, Method 2: NLT 10.0%• Microbial
Enumeration Tests : The total aerobic bacterial count
does not exceed 105 cfu/g, the total combined molds
and yeasts count does not exceed 103 cfu/g, and the
bile-tolerant Gram-negative bacteria count does not
exceed 103 cfu/g.
16. Conti..
Microbiological Procedures for Absence of Specified
Microorganisms : Meets the requirements of the tests
for absence of Salmonella species and Escherichia coli
Articles of Botanical Origin, Aflatoxins : Meets the
requirements
17. Conti..
ADDITIONAL REQUIREMENTS
Packaging and Storage: Preserve in well-closed
containers, protected from light and moisture, and store
at room temperature.
Labeling: The label states the Latin binomial and,
following the official name, the part of the plant
contained in the article.
18. 3. Fennel Oil
Fennel Oil is the volatile oil distilled with steam from
the dried, ripe fruit of Foeniculum vulgare Mill. (Fam.
Apiaceae).NOTE—If solid material has separated,
carefully warm the Fennel Oil until it is completely
liquefied, and mix before using.
Packaging and storage— Preserve in tight containers.
19. Conti…
Labeling— The label states the Latin binomial and,
following the official name, the part of the plant source
from which the article was derived. The label also
states that if solid material has separated, carefully
warm Oil until it is completely liquefied, and mix
before using.
20. Conti…
Solubility in 90 percent alcohol— One volume
dissolves in 1 volume of 90 percent alcohol.
Specific gravity : between 0.953 and 0.973.
Congealing temperature : not lower than 3.
Angular rotation: between +12 and +24.
Refractive index : between 1.528 and 1.538 at 20.
Heavy metals, Method II : 0.004%.
Residual solvents : meets the requirements.
21. 4. Powdered Turmeric
Extract
Powdered Turmeric Extract is prepared from the
pulverized rhizomes of Curcuma longa L. (Fam.
Zingiberaceae), using acetone, methanol, or other
suitable solvents. It contains NLT 20% of total
curcuminoids, calculated on the dried basis. It may
contain other added substances.
22. IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test
Standard solution: 0.2 mg/mL of USP Curcuminoids
RS in acetone
Sample solution: 10 mg/mL of Powdered Turmeric
Extract in acetone
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture, typically 20 cm long (TLC plates)
Application volume: 10 µL, as bands
Developing solvent system: Chloroform, methanol, and
formic acid (96:4:1)
Analysis
23. Conti..
Samples: Standard solution and Sample solution
Apply the samples as bands to a suitable thin-layer
chromatographic plate (see Chromatography 621, Thin
Layer Chromatography). Use a saturated chamber.
Develop the chromatograms until the solvent front has
moved up about three-fourths of the length of the plate.
Remove the plate from the chamber, dry, and examine
in daylight and under UV light at 365 nm.
.
24. Conti..
Acceptance criteria: The Sample solution
chromatogram shows yellowish-brown bands due to
bisdesmethoxycurcumin, desmethoxycurcumin, and
curcumin at RF values of about 0.4, 0.6, and 0.7,
respectively, corresponding in position and color to
those obtained from the Standard solution.
• B. The retention times of the peaks for curcumin,
desmethoxycurcumin, and bisdesmethoxycurcumin of
the Sample solution chromatogram correspond to those
of the Standard solution for the appropriate USP
Reference Standard, as obtained in the test for Content
of Curcuminoids
25. Conti..
CONTAMINANTS
• Heavy Metals, Method III 231: NMT 20 ppm
• Articles of Botanical Origin, General Method for
Pesticide Residues Analysis 561: Meets the
requirements
• Articles of Botanical Origin, Test for Aflatoxins 561:
Meets the requirements
• Microbial Enumeration Tests 2021: The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
combined molds and yeasts count does not exceed 103
cfu/g
26. Conti..
Microbiological Procedures for Absence of Specified
Microorganisms: Meets the requirements of the tests
for the absence of Salmonella species and Escherichia
coli
• Botanical Extracts, Residual Solvents: Meets the
requirements
SPECIFIC TESTS
• Loss on Drying: Dry 1.0 g at 105⁰C for 2 h: it loses
NMT 7.0% of its weight.
27. Conti..
ADDITIONAL REQUIREMENTS
• Packaging and Storage: Preserve in well-closed
containers. Protect from light and moisture, and store at
controlled room temperature.
• Labeling: The label states the Latin binomial and,
following the official name, the part of the plant from
which the article was prepared. It meets other labeling
requirements under Botanical Extracts 565
28. 5.Powdered Garlic
Extract
Garlic consists of the fresh or dried compound
bulbs of Allium sativum Linné (Fam. Liliaceae).
It contains not less than 0.5 percent of alliin and
not less than 0.2 percent of -glutamyl-(S)-allyl-L-
cysteine, calculated on the dried basis.
29. Conti…
Powdered Garlic Extract is prepared from fresh Garlic
bulbs by extraction with alcohol.
The ratio of the starting crude plant material to
Powdered Extract is between 9.5:1 and 13.5:1.
It contains not less than 4.0 percent of alliin
(C6H11NO3S).
It may contain added Powdered Garlic or other
suitable substances.
30. Conti..
Packaging and storage— Preserve in tight containers,
in a cool place, protected from light.
Labeling— The label states the Latin binomial and,
following the official name, the part of the plant from
which the article was prepared. The label also indicates
the content of alliin, the extracting solvent or solvent
mixture used for preparation, and the ratio of the
starting crude plant material to Powdered Extract. It
meets the requirements for Labeling under Botanical
Extracts
31. Botanic characteristics
Macroscopic—
Subglobular compound bulbs, 3 to 5 cm in width,
consisting of 8 to 20 cloves, the whole surrounded by 2
to 5 layers of white scale leaves attached to a flattened,
circular base; cloves ovoid and 3- to 4-sided, summit
acute, narrowed into a threadlike portion of fiber base,
truncate, each clove covered with a white scale leaf and
a pinkish white epidermis, easily separated from the
solid portion, consisting of two flaky scale leaves and
two yellowish green conduplicate foliage leaves.
32. Microscopic
The protective leaf contains an epidermis enclosing a
mesophyll free from chlorophyll.
The outer epidermis consists of lignified sclereid cells
of thick, pitted walls, elongated, covered with thin
cuticle, long fibers up to 500 µm in length and 30 µm
in width.
33. Conti..
The cortical cells are thick-walled, nonlignified,
tending to collapse on maturity, isodiametric, and
contain purple pigments.
The vascular bundles consist of lignified spiral and
annular vessels.
The storage leaves show an outer epidermis of thin,
delicate cells of variable shape, arranged in somewhat
irregular rows, 60 µm in length and 30 µm in width.
Stomata are present on the outer epidermis only at the
extreme tip near the base of the foliage leaves.
34. Conti..
The mesophyll consists of swollen storage parenchyma
cells filled with fine granular reserve material; scattered
in the cortex are about 20 laticiferous tubes, 500 to
1000 µm in length.
Two series of vascular bundles consisting of narrow
lignified spiral and annular vessels are arranged in the
mesophyll.
35. Identification
A: Thin-Layer Chromatographic Identification
Test 201—
Test solution— Cut a freeze-dried garlic bulb into small
pieces, transfer about 1 g of the cut pieces to an
extractor, and extract with two 20-mL portions of a
mixture of methanol and water (1:1), combining the
extracts. Concentrate to a small volume (about 5 mL),
using a rotary evaporator.
Standard solution A: 0.5 mg of USP L-Methionine
RS per mL.
36. Conti..
Standard solution B: 0.5 mg of USP Alliin RS per mL,
in a mixture of methanol and water (1:1).
Application volume: 20 µL, applied separately as 10-
mm bands.
Developing solvent system: a mixture of butyl
alcohol, n-propyl alcohol, glacial acetic acid, and
water (3:1:1:1).
37. Conti..
Procedure— Proceed as directed in the chapter. Spray with a 0.2
in 100 solution of ninhydrin in a mixture of butyl alcohol and 2
N acetic acid (95:5), heat at 100 to 105⁰c for about 10 minutes,
and immediately examine the plate.
The chromatogram of the Test solution shows many orange and
pinkish violet zones: a violet zone having an RF value of about
0.89; a pink zone having an RF value of about 0.5 and
corresponding in color and RF value to that obtained from the
chromatogram of Standard solution A; a pinkish zone having
an RF value of about 0.43; a strong orange zone having
an RF value of about 0.38; a pinkish violet zone having
an RF value of about 0.3 and corresponding in color
and RF value to that obtained from the chromatogram
of Standard solution B; and additional pinkish orange zones
situated very close to each other just below the zone attributed
to alliin in the chromatogram of Standard solution B.
38. Conti…
B: Transfer about 10 g of garlic bulbs that have been
cut into small pieces to a suitable flask. Add 10 mL of
1N sodium hydroxide and 10 mL of water, heat the
flask in boiling water for 10 minutes, cool, and filter.
Add a few drops of freshly prepared sodium
nitroferricyanide TS to 2 mL of the filtrate: appearance
of a red or orange-red color indicates the presence of
sulfur-containing compounds in the test specimen
39. Conti..
C: The retention time of the major peak in the
chromatogram of the Test solution corresponds to
that in the chromatogram of the Standard solution,
as obtained in the test for Content of alliin.
40. Conti…
Total ash : not more than 5.0%.
Acid-insoluble ash : not more than 1.0%.
Water content : not more than 65.0% for fresh bulbs,
and not more than 7.0% for dried bulbs.
Pesticide residues : meets the requirements.
