Ginkgo
201
Test for flavonoids
USP35
Extraction solvent:
Alcohol, hydrochloric acid, and water (25:4:10)
• Heavy Metals, Method III 231:
NMT 20 ppmUSP35
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.USP35
• Absence of Specified Microorganisms 2022:
Meets the requirements of the tests for absence of Salmonella species and Escherichia coliUSP35
731:
Dry 1.0 g of finely powdered Ginkgo at 105
for 2 h: it loses NMT 11.0% of its weight. USP35
11
USP Isorhamnetin RS
USP35
DEFINITION
Ginkgo consists of the dried leaf of Ginkgo biloba L. (Fam. Ginkgoaceae). It contains NLT 0.5% of flavonoids, calculated as flavonol glycosides, with a mean molecular mass of 756.7; and NLT 0.1% of terpene lactones, calculated as the sum of bilobalide (C15H18O8), ginkgolide A (C20H24O9), ginkgolide B (C20H24O10), and ginkgolide C (C20H24O11), both on the dried basis.
IDENTIFICATION
Change to read:
• Thin-Layer Chromatographic Identification Test 201
Test for flavonoids
Standard solution:
A solution of 0.6 mg/mL of USP Rutin RS, 0.2 mg/mL of USP Chlorogenic Acid RS, and 0.2 mg/mL of USP Quercetin RS in methanol
Sample solution:
Transfer 1.0 g of finely powdered Ginkgo to a 50-mL round-bottom flask fitted with a reflux condenser. Add 10 mL of methanol, reflux in a water bath for 10 min, allow to cool to room temperature, and filter. [Note—Reserve some of the Sample solution for use in the Test for terpene lactones. ]
Adsorbent:
Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)
Application volume:
5 µL
Developing solvent system:
Ethyl acetate, water, anhydrous formic acid, and glacial acetic acid (100:26:11:11)
Spray reagent 1:
5 mg/mL of 2-aminoethyl diphenylborinate in methanol
Spray reagent 2:
50 mg/mL of polyethylene glycol 400 in alcohol
Analysis
Samples:
Standard solution and Sample solution
Before development of the chromatograms, saturate the chamber for 20 min with Developing solvent system. Record temperature and humidity in the laboratory. If the relative humidity exceeds 50%, condition the plate to about 35% relative humidity using a suitable device. Apply the samples separately as bands to a suitable thin-layer chromatographic plate (see Chromatography 621), and allow the bands to dry. Develop the plate over a path of 6 cm, remove the plate from the chromatographic chamber, and dry in a circulating air oven at 105 for 5 min. Immediately spray the hot plate with Spray reagent 1, then with Spray reagent 2, dry, and examine under long-wavelength UV light.
621), and allow the bands to dry. Develop the plate over a path of 6 cm, remove the plate from the chromatographic chamber, and dry in a circulating air oven at 105 for 5 min. Immediately spray the hot plate with Spray reagent 1, then with Spray reagent 2, dry, and examine under long-wavelength UV light.
Acceptance criteria:
The Standard solution shows in its lower part with increasing RF values a yellowish-brown fluorescent zone due to rutin (RF 0.28), a light blue fluorescent zone due to chlorogenic acid (RF 0.36), and a yellow fluorescent zone due to quercetin (RF 0.92). The Sample solution shows a yellowish-brown fluorescent zone, a light blue fluorescent zone, and a yellowish-brown fluorescent zone at RF similar to those of rutin, chlorogenic acid, and quercetin, respectively, in the Standard solution. Additional yellowish to yellowish-green zones due to flavonoids detected in the Sample solution chromatogram include one zone below the rutin zone, two zones between the rutin and chlorogenic acid zones, and two zones above the chlorogenic acid zone. Other zones may be seen in the Sample solution chromatogram.
Test for terpene lactones
Standard solution:
Dissolve an amount of USP Ginkgo Terpene Lactones RS in methanol to obtain a solution containing in each mL about 1.0, 0.9, 0.6, 0.7, and 0.2 mg of bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, and ginkgolide J, respectively.
Sample solution:
Use the Sample solution prepared in the Test for flavonoids.
Adsorbent:
Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)
Application volume:
5 µL
Developing solvent system:
Toluene, ethyl acetate, acetone, and methanol (20 : 10 : 10 : 1.2)
Spray reagent:
Acetic anhydride
Analysis
Samples:
Standard solution and Sample solution
Immerse a suitable thin-layer chromatographic plate coated with Adsorbent for 2 s in a 8 g/200 mL solution of sodium acetate in methanol. Allow the excess liquid to drip from the plate, dry in a forced-air oven at 70 for 30 min, and cool in a desiccator. Apply the samples separately as bands to the impregnated plate (see Chromatography 621), and allow the spots to air-dry. Before development of the chromatograms, saturate the chamber for 20 min with Developing solvent system. Record temperature and humidity in the laboratory. If the relative humidity exceeds 50%, condition the plate to about 35% relative humidity, using a suitable device. Develop the chromatograms until the solvent front has moved up about 90% of the plate, remove the plate from the chromatographic chamber, and dry in cold air. Spray the plate with Spray reagent, heat at 180 for 10 min, cool, and examine under short-wavelength UV light.
for 30 min, and cool in a desiccator. Apply the samples separately as bands to the impregnated plate (see Chromatography 621), and allow the spots to air-dry. Before development of the chromatograms, saturate the chamber for 20 min with Developing solvent system. Record temperature and humidity in the laboratory. If the relative humidity exceeds 50%, condition the plate to about 35% relative humidity, using a suitable device. Develop the chromatograms until the solvent front has moved up about 90% of the plate, remove the plate from the chromatographic chamber, and dry in cold air. Spray the plate with Spray reagent, heat at 180 for 10 min, cool, and examine under short-wavelength UV light.
Acceptance criteria:
The Standard solution chromatogram shows five distinct quenching zones corresponding to the different ginkgo terpene lactones: ginkgolide C, ginkgolide J, ginkgolide B, ginkgolide A, and bilobalide at RF values of about 0.13, 0.18, 0.32, 0.38, and 0.45, respectively. The Sample solution chromatogram shows a strong quenching zone at the application position, a broad quenching zone near the solvent front, and five distinct quenching zones corresponding to different ginkgo terpene lactones at RF similar to those detected in the Standard solution chromatogram.
[Note—RF values may differ from one plate to another due to the impregnation step. ]
USP35COMPOSITION
Change to read:
• Content of Flavonol Glycosides
Extraction solvent:
Alcohol, hydrochloric acid, and water (25:4:10)
Mobile phase:
Methanol, water, and phosphoric acid (100:100:1)
Standard solution A:
0.02 mg/mL of USP Quercetin RS in methanol
Standard solution B:
0.02 mg/mL of USP Kaempferol RS in methanol
Standard solution C:
0.005 mg/mL of USP Isorhamnetin RS in methanol
Sample solution:
Transfer about 1.0 g of Ginkgo, finely powdered, to a 250-mL flask fitted with a reflux condenser. Add 78 mL of Extraction solvent, and reflux on a hot water bath for 135 min. [Note—The solution will turn deep red. The color of the solution is not a definitive indication of reaction completeness. ] Allow to cool at room temperature. Decant to a 100-mL volumetric flask. Add 20 mL of methanol to the 250-mL flask, and sonicate for 30 min. Filter, collect the filtrate in the 100-mL volumetric flask, wash the residue on the filter with methanol, collect the washing in the same 100-mL volumetric flask, dilute with methanol to volume, and mix.
Chromatographic system
Mode:
LC
Detector:
UV 370 nm
Column:
4.6-mm × 25-cm; packing L1
Flow rate:
1.5 mL/min
Injection size:
20 µL
System suitability
Samples:
Standard solution A, Standard solution B, and Standard solution C
[Note—The relative retention times for quercetin, kaempferol, and isorhamnetin are about 1.0, 1.8, and 2.0, respectively; Standard solution A, Standard solution B, and Standard solution C. ]
Suitability requirements
Relative standard deviation:
NMT 2.0% determined from the quercetin peak in repeated injections, Standard solution A
Analysis
Samples:
Standard solution A, Standard solution B, Standard solution C, and Sample solution
Calculate the percentage of each flavonol glycoside in the portion of Ginkgo taken:
Result = (rU/rS) × (CS/W) × F × 10
| rU | = | = peak area of the relevant analyte from the Sample solution |
| rS | = | = peak area of the relevant analyte from Standard solution A, Standard solution B, or Standard solution C |
| CS | = | = concentration of the relevant analyte in Standard solution A, Standard solution B, or Standard solution C (mg/mL) |
| W | = | = weight of Ginkgo taken to prepare the Sample solution (g) |
| F | = | = mean molecular mass factor to convert each analyte into flavonol glycoside with a mean molecular mass of 756.7: 2.504 for quercetin, 2.437 for isorhamnetin, and 2.588 for kaempferol |
Calculate the total percentage of flavonol glycosides by adding the individual percentages calculated.
Acceptance criteria:
NLT 0.5% of flavonoids, as flavonol glycosides, with a mean molecular mass of 756.7, on the dried basisUSP35
USP35
• Content of Terpene Lactones
Solvent:
Methanol and water (9:1)
Buffer solution:
Dissolve 1.19 g of dibasic sodium phosphate and 8.25 g of monobasic potassium phosphate in 1000 mL of water, and adjust to a pH of 5.8.
Diluent:
Methanol and water (1:1)
Solution A:
Water
Solution B:
Methanol
Mobile phase:
See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 75 | 25 |
| 23 | 52 | 48 |
| 28 | 52 | 48 |
| 30 | 25 | 75 |
| 35 | 10 | 90 |
| 40 | 75 | 25 |
| 50 | 75 | 25 |
Standard solutions:
Dissolve accurately weighed quantities of USP Ginkgo Terpene Lactones RS in Diluent, sonicate for a few min, and dilute with Diluent to obtain solutions with known concentrations of about 0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL. Pass through a filter of 0.45-µm or finer pore size.
Sample solution:
Transfer about 2.5 g of Ginkgo, accurately weighed, to a 30-mL glass centrifuge tube with a screw cap and PTFE gasket. Add 10.0 mL of Solvent, seal the tube, and mix well on a vortex mixer. Heat in a water bath at 90 for 30 min. Mix the hot suspension on a vortex mixer, and repeat the heating at 90 for 30 min. Cool, centrifuge, transfer the supernatant to a flask, and return the residue to the glass tube. Repeat the extraction two more times, each time using 10.0 mL of Solvent. Combine the extracts, allow them to cool to room temperature, and evaporate to dryness under vacuum in a water bath maintained at 50. Add 10 mL of Buffer solution to the residue, and sonicate for 5 min. Quantitatively transfer the solution to a glass chromatographic tube filled with chromatographic siliceous earth capable of holding 20 mL of aqueous phase.1 Rinse the beaker with two 5-mL portions of Buffer solution, and transfer the washings to the column. [Note—Do not exceed 20 mL of total aqueous phase or the holding capacity of the chromatographic tube. ]
for 30 min. Mix the hot suspension on a vortex mixer, and repeat the heating at 90 for 30 min. Cool, centrifuge, transfer the supernatant to a flask, and return the residue to the glass tube. Repeat the extraction two more times, each time using 10.0 mL of Solvent. Combine the extracts, allow them to cool to room temperature, and evaporate to dryness under vacuum in a water bath maintained at 50. Add 10 mL of Buffer solution to the residue, and sonicate for 5 min. Quantitatively transfer the solution to a glass chromatographic tube filled with chromatographic siliceous earth capable of holding 20 mL of aqueous phase.1 Rinse the beaker with two 5-mL portions of Buffer solution, and transfer the washings to the column. [Note—Do not exceed 20 mL of total aqueous phase or the holding capacity of the chromatographic tube. ]
Allow the Buffer solution to be absorbed into the column. After 15 min, elute the column with 100 mL of ethyl acetate, collect the ethyl acetate solution, and evaporate to dryness under vacuum in a water bath maintained at 50. Dissolve the residue in 10.0 mL of Diluent.
. Dissolve the residue in 10.0 mL of Diluent.
Chromatographic system
Mode:
LC
Detector:
Evaporative light-scattering detector. [Note—The parameters of the detector are adjusted to achieve the best signal-to-noise ratio, according to manufacturer recommendations. ]
Column:
4.6-mm × 25-cm; packing L1
Column temperature:
25 ± 1
Flow rate:
1 mL/min
Injection size:
15 µL
System suitability
Samples:
Standard solutions
Suitability requirements
Chromatogram similarity:
The chromatograms from the Standard solutions are similar to the reference chromatogram provided with the lot of USP Ginkgo Terpene Lactones RS being used.
Relative standard deviation:
NMT 2.0% determined from the bilobalide peak in repeated injections
Correlation coefficient:
NLT 0.995 for the regression line as determined in the Analysis
Analysis
Samples:
Standard solutions and Sample solution
Record the chromatograms, and identify the peaks of the relevant analytes in the chromatograms of the Standard solutions by comparison with the reference chromatogram being used. Measure the areas of the analyte peaks. Plot the logarithms of the relevant peak responses versus the logarithms of concentrations, in mg/mL, of each analyte from the Standard solutions, and determine the regression line using a least-squares analysis.
From the graphs, determine the concentration, C, in mg/mL, of the relevant analyte in the Sample solution.
Separately calculate the percentages of bilobalide (C15H18O8), ginkgolide A (C20H24O9), ginkgolide B (C20H24O10), and ginkgolide C (C20H24O11) in the portion of Ginkgo taken:
Result = 1000 × (C/W)
| C | = | = concentration of the relevant analyte in the Sample solution (mg/mL) |
| W | = | = weight of Ginkgo taken to prepare the Sample solution (mg) |
Calculate the total percentage of terpene lactones in the portion of Ginkgo taken by adding the percentages calculated for each analyte.
Acceptance criteria:
NLT 0.1% of terpene lactones, calculated as the sum of bilobalide, ginkgolide A, ginkgolide B, and ginkgolide C, on the dried basis
CONTAMINANTS
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561:
Meets the requirements
561:
Meets the requirements
Add the following:
• Heavy Metals, Method III 231:
NMT 20 ppmUSP35
Change to read:
• 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.USP35
Add the following:
• Absence of Specified Microorganisms 2022:
Meets the requirements of the tests for absence of Salmonella species and Escherichia coliUSP35
SPECIFIC TESTS
• Botanic Characteristics
Macroscopic:
Dried whole, folded, or fragmented leaves, with or without attached petiole, vary from khaki green to greenish brown in color, are often more brown at the apical edge, and darker on the adaxial surface. Lamina are broadly obcuneate (fan-shaped), 2–12 cm in width and 2–9.5 cm in length from petiole to apical margin; mostly 1.5–2 times wider than long. The base margins are entire, concave; apical margin sinuate, usually truncate or centrally cleft, and rarely multiply cleft. The surface is glabrous, with wrinkled appearance due to prominent dichotomous venation appearing parallel and extending from the lamina base to the apical margin. Petioles, similar color to leaf, are channeled on the adaxial surface, and 2–8 cm in length.
Histology
Transverse section of lamina:
A thin but marked cuticle occurs over a single layer of epidermal cells on both surfaces. Stomata are present on the lower surface only, with guard cells sunken with respect to adjacent epidermal cells. Palisade elements, elongated, at right angles to the surface and often irregular in appearance, occur just below the upper epidermis. Vascular bundles occur at intervals along the width of the blade, with adjacent cluster crystals of calcium oxalate. Cells of the mesophyll are smaller than the palisade cells, elongated, parallel to the leaf surface, and separated by large intercellular spaces.
Powdered lamina and petiole:
Under the microscope, transverse fragments of the leaf display a smooth cuticle, present on both leaf surfaces and staining pinkish orange with sudan III TS. In surface view, cells of the upper epidermis are elongated and wavy-walled, with abundant yellow droplets 2–12 µm in diameter visible in mature and old leaves but not in young leaves. Cells of the lower epidermis are similar in shape but have straighter walls and are interrupted by anisocytic stomata. Numerous lignified elements derived from the lamina and petiole are present, including xylem vessels with annular thickening, tracheids, and vessels with bordered pits. The extent of lignification, particularly in the petiole, increases with age of leaf. Calcium oxalate crystals are numerous, present scattered or associated with vessels, ranging in size from 5–50 µm in young leaves and 15–100 µm in mature leaves. Under crossed polaroids, numerous smaller prism- or tear-shaped shiny features of indeterminate nature may be present. Very occasional, highly elongated, uniseriate, covering trichomes with no obvious cross walls and smooth or warty surfaces may be seen. Mature leaves may show the presence of very rare, polygonal to circular starch granules approximately 20 µm in diameter, with a central hilum and exhibiting a marked Maltese cross under crossed polaroids.
• Articles of Botanical Origin, Foreign Organic Matter 561:
NMT 3.0% of stems and NMT 2.0% of other foreign organic matter
561:
NMT 3.0% of stems and NMT 2.0% of other foreign organic matter
Change to read:
• Loss on Drying 731:
Dry 1.0 g of finely powdered Ginkgo at 105 for 2 h: it loses NMT 11.0% of its weight. USP35
• Articles of Botanical Origin, Total Ash 561:
NMT 11.0%, determined on 1.0 g of finely powdered Ginkgo
561:
NMT 11.0%, determined on 1.0 g of finely powdered Ginkgo
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.
Change to read:
• USP Reference Standards 11
USP Chlorogenic Acid RS 
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USP Ginkgo Terpene Lactones RS
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USP Isorhamnetin RS
USP Kaempferol RS
USP35
USP Quercetin RS
USP Rutin RS
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1
Suitable commercially available material is Extrelut® NT 20 from E Merck Science.
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 1323
Pharmacopeial Forum: Volume No. 36(6) Page 1602