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Printed by: USP NF Official Date: Official as of 01-Dec-2015 Document Type: DIETARY SUPPLEMENTS @2024 USPC
Do Not Distribute DOI Ref: r40b6 DOI: [Link]
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of methanol, heat gently for 15–20 min, dilute with
Andrographis acetonitrile to volume, and mix. Before injection, pass
DEFINITION through a membrane filter of 0.45-µm or finer pore size,
Andrographis consists of the dried stems and leaves of discarding the first 5 mL of the filtrate.
Andrographis paniculata (Burm. f.) Nees (Fam. Acanthaceae). Sample stock solution: Transfer about 2.0 g of finely
It contains NLT 1.0% of diterpene lactones, calculated on the powdered Andrographis to a 250-mL flask fitted with a
dried basis as the sum of andrographolide, reflux condenser. Add 50 mL of methanol, reflux for 15 min,
neoandrographolide, 14-deoxy- cool to room temperature, and decant the supernatant.
11,12-didehydroandrographolide, and andrograpanin. Repeat until the extract is colorless. Combine the extracts,
filter, concentrate under vacuum, and adjust the volume to
IDENTIFICATION 50.0 mL using methanol.
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Sample solution: Transfer 25.0 mL of Sample stock solution
á201ñ to a 50-mL volumetric flask, dilute with acetonitrile to
Standard solution 1: Use Standard solution A, prepared as volume, and mix. Before injection, pass through a
directed in the test for Content of Diterpene Lactones. membrane filter of 0.45-µm or finer pore size, discarding
Standard solution 2: Sonicate an amount of USP Powdered the first 5 mL of the filtrate.
Andrographis Extract RS, equivalent to about 15 mg of Mobile phase: See Table 1.
diterpene lactones, for 10–15 min in 25 mL of methanol,
centrifuge, and use the supernatant. Table 1
Sample solution: Use Sample stock solution, prepared as Time Solution A Solution B
directed in the test for Content of Diterpene Lactones. (min) (%) (%)
Adsorbent: Chromatographic silica gel mixture with an
0 95 5
al
average particle size of 10–15 µm (TLC plates)
Application volume: 10 µL, as 5–10 mm bands 18 55 45
Developing solvent system: Chloroform, acetone, and 25 20 80
toluene (2:2:1)
Derivatization reagent: A mixture of 1% vanillin in alcohol 28 20 80
and 10% sulfuric acid in alcohol (1:1)
Analysis
Samples: Standard solution 1, Standard solution 2, and
Sample solution
ci 35
40
45
55
95
95
45
5
Use a saturated chamber. Develop until the solvent front
has moved up about 90% of the length of the plate.
ffi
Remove the plate from the chamber, dry, treat with Chromatographic system
Derivatization reagent, heat for 5–10 min at 100°, and (See Chromatography á621ñ, System Suitability.)
examine under white light. Mode: LC
Acceptance criteria: The Sample solution exhibits three Detector: UV 223 nm
main grayish-blue zones with RF values of approximately Column: 4.6-mm × 25-cm; 5-µm packing L1
0.4, 0.6, and 0.8 that correspond in position and color to Flow rate: 1.5 mL/min
O
zones in Standard solution 2. Standard solution 1 exhibits a Injection volume: 20 µL
grayish-blue zone due to andrographolide at an RF of about System suitability
Samples: Standard solution A and Standard solution B
0.4. The Sample solution exhibits a zone similar in color and
Suitability requirements
RF value to that due to andrographolide in Standard solution
The chromatogram of Standard solution B is similar to the
1. reference chromatogram provided with the lot of USP
• B. The retention time of the main peak of the Sample Powdered Andrographis Extract RS being used.
solution obtained in the test for Content of Diterpene Column efficiency: NLT 5000 theoretical plates,
Lactones corresponds to that of andrographolide in Standard solution A
Standard solution A. Identify other diterpene lactone peaks Tailing factor: NMT 1.5 for the andrographolide peak,
in the Sample solution by comparison with Standard solution Standard solution A
B and the reference chromatogram provided with the lot of Relative standard deviation: NMT 2.0%, determined
USP Powdered Andrographis Extract RS being used. The for the andrographolide peak in replicate injections,
Sample solution shows additional peaks corresponding to Standard solution A
neoandrographolide, 14-deoxy- Resolution: NLT 5 between the neoandrographolide and
11,12-didehydroandrographolide, and andrograpanin. 14-deoxy-11,12-didehydroandrographolide peaks,
COMPOSITION Standard solution B
• CONTENT OF DITERPENE LACTONES Analysis
Solution A: Dissolve 0.14 g of potassium dihydrogen Samples: Standard solution A, Standard solution B, and
phosphate in 900 mL of water, add 0.5 mL of phosphoric Sample solution
acid, dilute with water to 1000 mL, mix, filter, and degas. Using the chromatogram of Standard solution A, Standard
Solution B: Acetonitrile, filtered and degassed solution B, and the reference chromatogram provided
Standard solution A: Dissolve a weighed quantity of USP with the lot of USP Powdered Andrographis Extract RS
Andrographolide RS in methanol to obtain a 1.0-mg/mL being used, identify the retention times of the peaks
solution. Transfer 5.0 mL of this solution to a 10-mL corresponding to the different diterpene lactones. The
volumetric flask, dilute with acetonitrile to volume, approximate relative retention times of the different
and mix. diterpene lactones are provided in Table 2.
Standard solution B: Transfer an amount of USP Powdered
Andrographis Extract RS, equivalent to about 25 mg of
diterpene lactones, to a 50-mL volumetric flask, add 25 mL
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Printed on: Fri Jan 05 2024, 10:31:24 PM(EST) Status: Currently Official on 06-Jan-2024 DocId: GUID-CF2561BF-722D-4BAF-AE59-D693D327B577_1_en-US
Printed by: USP NF Official Date: Official as of 01-Dec-2015 Document Type: DIETARY SUPPLEMENTS @2024 USPC
Do Not Distribute DOI Ref: r40b6 DOI: [Link]
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Table 2 cuneate-decurrent base, margin entire or undulate; the
Relative upper surface is green, the lower surface grayish-green;
Retention both surfaces are glabrous. Pharmacopeial article consists
Analyte Time of dry mixtures of crisp, dark-green broken leaves and
Andrographolide 1.00 quadrangular stems; leaves are brittle; stems are fracture
short, fibrous.
Neoandrographolide 1.16 Microscopic
14-Deoxy-11,12-didehydroandrographolide 1.31 Transverse section of stems: Epidermal layer shows cells
containing round, long-elliptical or clavate calcium
Andrograpanin 1.50 carbonate deposits (cystoliths), 1–4 celled nonglandular
hairs and multicellular, disk-shaped glandular hairs;
Separately calculate the percentages of andrographolide, collenchyma is below the epidermis and in the bulges;
neoandrographolide, 14-deoxy- endodermis is distinct; vascular bundles surround the
11,12-didehydroandrographolide, and andrograpanin in parenchyma of the central pith; small acicular crystals of
the portion of Andrographis taken: calcium oxalate are present in the cortex and pith.
Transverse section of leaves: Subsquare or rectangular
Result = (rU/rS) × (CS/W) × 10F upper and lower epidermal cells; lower epidermal cells are
relatively smaller; both epidermal layers show cells
rU = peak area of each identified diterpene lactone in containing cystoliths, nonglandular hairs and glandular
the Sample solution hairs similar to those of the stem; mesophyll is composed
rS = peak area of andrographolide in Standard of 1–2 layers of palisade parenchyma and spongy
solution A parenchyma; loosely arranged spongy parenchyma
CS = concentration of USP Andrographolide RS in appear across the upper part of the midrib; vascular
al
Standard solution A (mg/mL) bundles of midrib are collateral and grooved; cells
W = weight of Andrographis taken to prepare the containing cystoliths appear above the xylem.
Sample solution (g) • LOSS ON DRYING á731ñ
F = conversion factor: 1.00 for andrographolide, Sample: 1.0 g of finely powdered Andrographis
3.90 for neoandrographolide, 1.45 for 14-deoxy- Analysis: Dry the Sample at 105° for 3 h.
11,12-didehydroandrographolide, and 2.65 for
andrograpanin
ci Acceptance criteria: NMT 12.0%
• ARTICLES OF BOTANICAL ORIGIN, Total Ash á561ñ
Sample: 1.0 g of finely powdered Andrographis
Acceptance criteria: NLT 1.0% for the sum of the Acceptance criteria: NMT 15%
percentages of andrographolide, neoandrographolide, • ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash á561ñ:
ffi
14-deoxy-11,12-didehydroandrographolide, and NMT 3.0%
andrograpanin, on the dried basis • ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
IMPURITIES Method 2 á561ñ: NLT 8.0%
• ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental • MICROBIAL ENUMERATION TESTS á2021ñ: The total aerobic
Impurities á561ñ: Meets the requirements bacterial count does not exceed 105 cfu/g; the total
• ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter combined molds and yeasts count does not exceed 103 cfu/
O
á561ñ: NMT 2.0% g; and the bile-tolerant Gram-negative bacterial count does
• ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis not exceed 103 cfu/g.
á561ñ: Meets the requirements • ABSENCE OF SPECIFIED MICROORGANISMS á2022ñ: Meets the
requirements of the tests for absence of Salmonella species
SPECIFIC TESTS and Escherichia coli
• BOTANICAL CHARACTERISTICS
Macroscopic: Stem is dark green, woody, 2–6 mm in ADDITIONAL REQUIREMENTS
diameter, bearing numerous branches, showing slightly • PACKAGING AND STORAGE: Preserve in well-closed
swollen nodes, the upper part is distinctly quadrangular containers, protected from light and moisture, and store at
with four bulges in the four corners, and the lower part is room temperature.
somewhat rounded; texture is fragile, easily broken; • LABELING: The label states the Latin binomial and, following
branches are quadrangular, often narrowly winged in the the official name, the parts of the plant contained in the
upper part. Leaves are simple, opposite, short, petiolated or article.
nearly sessile; lamina is crumpled and easily broken, • USP REFERENCE STANDARDS á11ñ
lanceolate or ovate-lanceolate when whole, 2–7 cm long, USP Andrographolide RS
1–3 cm wide, with acuminate apex, reticulate venation, and USP Powdered Andrographis Extract RS
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