Powdered Andrographis
DEFINITION
Powdered Andrographis is Andrographis reduced to a fine or very fine powder.
IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test 
201
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 10–15 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 10–15 µm (TLC plates)
Application volume:
10 µL, as 5–10 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)
Analysis
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 5–10 min at 100
, and examine under visible light.
, 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 the main zones of 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.
COMPOSITION
• 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 15–20 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 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.
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 95 | 5 |
| 18 | 55 | 45 |
| 25 | 20 | 80 |
| 28 | 20 | 80 |
| 35 | 55 | 45 |
| 40 | 95 | 5 |
| 45 | 95 | 5 |
Chromatographic system
Mode:
LC
Detector:
UV 223 nm
Column:
4.6-mm × 25-cm; 5-µm packing L1
Flow rate:
1.5 mL/min
Injection size:
20 µL
System suitability
Samples:
Standard solution A and Standard solution B
Suitability requirements
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
Analysis
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:
| Analyte | Relative Retention Time |
|---|---|
| Andrographolide | 1.00 |
| Neoandrographolide | 1.16 |
| 14-Deoxy-11,12-didehydroandrographolide | 1.31 |
| Andrograpanin | 1.50 |
Separately calculate the percentages of andrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and andrograpanin in the portion of Powdered Andrographis taken:
Result = (CS/W) × (rU/rS) × 10F
| rU | = | = peak response for each diterpene lactone from the Sample solution |
| rS | = | = peak response for andrographolide from Standard solution A |
| CS | = | = concentration of USP Andrographolide RS in Standard solution A (mg/mL) |
| W | = | = weight of Powdered Andrographis taken to prepare the Sample solution (g) |
| F | = | = conversion factor for each analyte (1.00 for andrographolide, 3.90 for neoandrographolide, 1.45 for 14-deoxy-11,12-didehydroandrographolide, and 2.65 for andrograpanin) |
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
IMPURITIES
Inorganic Impurities
• Heavy Metals, Method II 231:
NMT 20 ppm
231:
NMT 20 ppm
Organic Impurities
• Procedure: Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561:
Meets the requirements
561:
Meets the requirements
SPECIFIC TESTS
• Botanic Characteristics
Macroscopic:
It is a grayish brown powder.
Histology
Microscopic examination:
It reveals cells of the upper and lower epidermis of the leaves, some cells containing large cystoliths, up to 36 µm in diameter and 180 µm long, with a hilum-shaped scar in the large end; 1–4 celled nonglandular hairs; disk-shaped glandular hairs, 8-celled head and very short stalk; diacytic stomata mostly on the lower epidermis; stem epidermal cells, some cells containing cystoliths, stomata, nonglandular hairs and glandular hairs similar to those of the leaves; thin-walled parenchyma cells; collenchyma cells; acicular phloem fibers; tracheids; vessels, with spiral and scalariform thickening.
• Loss on Drying 731:
Dry 1.0 g of Powdered Andrographis at 105 for 3 h: it loses NMT 12.0% of its weight.
731:
Dry 1.0 g of 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 Powdered Andrographis
561:
NMT 15%, determined on 1.0 g of 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.
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
2022:
Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
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 parts of the plant contained in the article.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Maged H. Sharaf, Ph.D.
Principal Scientific Liaison 1-301-816-8318 |
(DS2010) Monographs - Dietary Supplements |
| 2021 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 2022 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1189
Pharmacopeial Forum: Volume No. 35(5) Page 1184