Andrographis consists of the dried stems and leaves of Andrographis paniculata Nees. (Fam. Acanthaceae). It contains NLT 1.0% of diterpene lactones, calculated on the dried basis as the sum of andrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide and andrograpanin.
• A. Thin-Layer Chromatographic Identification Test 201
Standard solution 1: Use Standard solution A, prepared as directed in the test for Content of Diterpene Lactones.
Standard solution 2: Sonicate an amount of USP Powdered Andrographis Extract RS, equivalent to about 15 mg of diterpene lactones, for 1015 min in 25 mL of methanol, centrifuge, and use the supernatant.
Sample solution: Use Sample stock solution, prepared as directed in the test for Content of Diterpene Lactones.
Adsorbent: Chromatographic silica gel mixture with an average particle size of 1015 µm (TLC plates)
Application volume: 10 µL, as 510 mm bands
Developing solvent system: Chloroform, acetone, and toluene (2:2:1)
Spray reagent: A mixture of 1% vanillin in alcohol and 10% sulfuric acid in alcohol (1:1)
Samples: Standard solution 1, Standard solution 2, and Sample solution
Use a saturated chamber. Develop the chromatograms until the solvent front has moved up about 90% of the length of the plate. Remove the plate from the chamber, dry, spray with Spray reagent, heat for 510 min at 100, and examine under visible light.
Acceptance criteria: The Sample solution exhibits three main grayish blue zones with RF values of approximately 0.4, 0.6, and 0.8 that correspond in position and color to zones in Standard solution 2. Standard solution 1 exhibits a grayish blue zone due to andrographolide at an RF of about 0.4. The Sample solution exhibits a zone similar in color and RF value to that due to andrographolide in Standard solution 1.
• B. The retention time of the main peak of the Sample solution obtained in the test for Content of Diterpene Lactones corresponds to that of andrographolide in Standard solution A. Identify other diterpene lactone peaks in the Sample solution by comparison with Standard solution B and the reference chromatogram provided with the lot of USP Powdered Andrographis Extract RS. The Sample solution shows additional peaks corresponding to neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and andrograpanin.
• Content of Diterpene Lactones
Solution A: Dissolve 0.14 g of potassium dihydrogen phosphate in 900 mL of water, add 0.5 mL of phosphoric acid, dilute with water to 1000 mL, mix, filter, and degas.
Solution B: Use filtered and degassed acetonitrile.
Standard solution A: Dissolve a weighed quantity of USP Andrographolide RS in methanol to obtain a solution having a known concentration of about 1.0 mg/mL. Transfer 5.0 mL of this solution to a 10-mL volumetric flask, dilute with acetonitrile to volume, and mix.
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 of methanol, heat gently for 1520 min, dilute with acetonitrile to volume, and mix. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first 5 mL of the filtrate.
Sample stock solution: Transfer about 2.0 g of finely powdered Andrographis to a 250-mL flask fitted with a reflux condenser. Add 50 mL of methanol, reflux on a water bath for 15 min, cool to room temperature, and decant the supernatant. Repeat until the last extract is colorless. Combine the extracts, filter, concentrate under vacuum, and adjust the volume to 50.0 mL using methanol.
Sample solution: Transfer 25.0 mL of Sample stock solution to a 50-mL volumetric flask, dilute with acetonitrile to volume, and mix. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first 5 mL of the filtrate.
Mobile phase: See the gradient table below.
Detector: UV 223 nm
Column: 4.6-mm × 25-cm; 5-µm packing L1
Flow rate: 1.5 mL/min
Injection size: 20 µL
Samples: Standard solution A and Standard solution B
The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Andrographis Extract RS.
Column efficiency: NLT 5000 theoretical plates, Standard solution A
Tailing factor: NMT 1.5 for the andrographolide peak, Standard solution A
Relative standard deviation: NMT 2.0%, determined from the andrographolide peak for replicate injections, Standard solution A
Resolution: NLT 5 between the neoandrographolide and 14-deoxy-11,12-didehydroandrographolide peaks, Standard solution B
Samples: Standard solution A, Standard solution B, and Sample solution
Using the chromatogram of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Powdered Andrographis Extract RS being used, identify the retention times of the peaks corresponding to the different diterpene lactones. The approximate relative retention times of the different diterpene lactones are provided in the following table:
Separately calculate the percentages of andrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and andrograpanin in the portion of Andrographis taken:
Result = (CS/W) × (rU/rS) × 10F
Acceptance criteria: NLT 1.0%, on the dried basis, of the sum of the percentages of andrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and andrograpanin
• Heavy Metals, Method II 231: NMT 20 ppm
• Procedure 1: Articles of Botanical Origin, Foreign Organic Matter 561: NMT 2.0%
• Procedure 2: Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561: Meets the requirements
• Botanic Characteristics
Macroscopic: Stem is dark green, woody, 26 mm in diameter, bearing numerous branches, showing slightly swollen nodes, the upper part is distinctly quadrangular with four bulges in the four corners, and the lower part is somewhat rounded; texture fragile, easily broken; branches quadrangular, often narrowly winged in the upper part. Leaves are simple, opposite, short petiolated or nearly sessile; lamina crumpled and easily broken, when whole, lanceolate or ovate-lanceolate, 27 cm long, 13 cm wide, with acuminate apex, reticulate venation and cuneate-decurrent base, margin entire or undulate; the upper surface green, the lower surface grayish green; both surfaces are glabrous. Pharmacopeial article consists of dry mixtures of crisp, dark-green broken leaves and quadrangular stems; leaves brittle; stems fracture short, fibrous.
Transverse section of stems: Epidermal layer showing cells containing round, long-elliptical or clavate calcium carbonate deposits (cystoliths), 14 celled nonglandular hairs and multicellular, disk-shaped glandular hairs; collenchyma below the epidermis and in the bulges; endodermis is distinct; vascular bundles surround the parenchyma of the central pith; small acicular crystals of calcium oxalate present in the cortex and pith.
Transverse section of leaves: Subsquare or rectangular upper and lower epidermal cells; lower epidermal cells are relatively smaller; both epidermal layers show cells containing cystoliths, nonglandular hairs and glandular hairs similar to those of the stem; mesophyll composed of 12 layers of palisade parenchyma and spongy parenchyma; loosely arranged spongy parenchyma appear across the upper part of the midrib; vascular bundles of midrib are collateral and grooved; cells containing cystoliths appear above the xylem.
• Loss on Drying 731: Dry 1.0 g of finely powdered Andrographis at 105 for 3 h: it loses NMT 12.0% of its weight.
• Articles of Botanical Origin, Total Ash 561: NMT 15%, determined on 1.0 g of finely powdered Andrographis
• Microbial Enumeration Tests 2021: 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 does not exceed 103 cfu/g.
• Microbiological Procedures for Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
• 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 parts of the plant contained in the article.
• USP Reference Standards 11
USP Andrographolide RS
USP Powdered Andrographis Extract RS
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USP35NF30 Page 1187Pharmacopeial Forum: Volume No. 35(5) Page 1183