Development of the U.S. Pharmacopeia’s Monograph on Ginkgo Biloba: Appendix

Ginkgo Leaf

Ginkgo leaf consists of the dried leaf of Ginkgo biloba Linn (Fam. Ginkgoaceae.

Packaging and storage

Preserve in well-closed containers, protected from light and moisture.

Labeling

The label states that it is Ginkgo Leaf and states also the genus and species.

USP Reference standards

USP Quercetin RS, USP Rutin RS.

Botanic characteristics

Macroscopic: Dried whole, folded or fragmented leaves, with or without attached petiole, varying from khaki green to greenish-brown in color, often more brown at the apical edge, and darker on the adaxial surface. Lamina broadly obcuneate (fan shaped), 2 to 12cm in width and 2 to 9.5cm in length from petiole to apical margin; mostly 1.5 to 2 times wider than long. Base margins entire, concave; apical margin sinuate, usually truncate or centrally cleft, and rarely multiple cleft. Surface glabrous, with wrinkled appearance due to prominent dichotomous venation appearing parallel and extending from the lamina base to the apical margin. Petiole of a similar color to leaf, channeled on the adaxial surface, 2 to 8cm in length.

HistologyTransverse 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 to 12µm 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 to 50µm in young leaves to 15 to 100µm 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 20µm in diameter with a central hilum and exhibiting a marked maltese cross under crossed polaroids.

Identification

A: Transfer 0.2g of finely powdered Ginkgo Leaf to a test tube, add 10mL of methanol, and heat on a water bath at 65° for 10 minutes. Shake the mixture frequently during the heating. Allow to cool to room temperature, filter, concentrate the filtrate on a hot water bath at 60° to half its volume, and cool. Apply separately, as bands, 20µL each of the test solution and a Standard solution of USP Rutin TS and a chlorogenic acid in methanol containing about 0.6mg per mL and 0.2mg per mL, respectively, to a suitable thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel, and allow the bands to dry. Develop the chromatograms in a mixture of ethyl acetate, water, anhydrous formic acid, and glacial acetic acid (67.5:17.5:7.5:7.5) until the solvent front has moved about 10 cm from the origin. Remove the plate from the chromatographic chamber, and dry it in a circulating air oven at 100° to 105°. Immediately spray the hot plate with a solution of diphenyl boryloxyethylamine in methanol containing 10mg per mL, then spray with a solution of polyethylene glycol 400 in alcohol containing 50mg per mL. Allow the plate to cool for 30 minutes, and examine it under long-wavelength ultraviolet light. The chromatogram of the Standard solution shows in its middle part, with increasing Rf values, the yellowish brown fluorescent zone due to rutin and a light blue fluorescent zone due to chlorogenic acid as well as two greenish brown-yellow fluorescent zones, located above. Other, less intense zones may be seen in the chromatogram of the test solution.

B: Prepare a suitable thin-layer chromatographic plate coated with a 0.50-mm layer of chromatographic silica gel as follows. Immerse the plate for 20 seconds in a solution of sodium acetate in methanol containing 1g per 10mL. Allow the excess coating liquid to drip from the plate, and dry in a forced-air oven at 70° for 30 minutes. Cool in a dessicator. Transfer 0.8g of the dried test specimen retained from the test for Loss on drying to a suitable flask fitted with a reflux condenser, add 5mL of a mixture of methanol and water (1 in 10), and heat under reflux for 15 minutes. While still hot, filter the contents of the flask with the aid of vacuum. Rinse the flask and the test specimen with 2mL of a mixture of methanol and water (2 in 100), and transfer the rinsings to the filter with the aid of vacuum. Return the powdered Ginkgo Leaf to the flask, add 4mL of a mixture of methanol and water (1 in 10), and repeat the extraction. After filtration, wash the residue of powdered Ginkgo Leaf twice with 1.5mL of a mixture of methanol and water (2 in 100). Combine the filtrates, and transfer the combined filtrates (about 12mL) to a solid-phase extraction column containing LI packing with a sorbent mass-to-column volume ratio of 1000mg per 3mL or equivalent. [NOTE — Initially pass 10mL of methanol and then 10mL of a mixture of methanol and water (2 in 100) through the column to condition it, Do not allow the column to dry.] Collect the eluate at the rate of 1 drop per second. Evaporate the eluate to dryness, and dissolve the residue in 2mL of methanol. Separately apply several 10-µL spots of the test solution to the impregnated plate, and allow the spots to air-dry. Develop the plate in a mixture of ethyl acetate and methyl acetate (1:1) in a chromatographic chamber without filter paper attached to the walls until the solvent front travels about three-fourths of the length of the plate. Remove the plate from the chamber, mark the solvent front, and dry in an oven at 105° for 15 minutes. Spray the plate with acetic anhydride, and heat in an oven at 140° for 25 minutes. Cool, and examine the plate under short- and long-wavelength ultraviolet light. [NOTE — The compounds present in high concentrations may be visible in daylight as light brown spots.] The presence of terpene lactones in the test solution is shown by the following spots detected in the chromatogram at both the short and long wavelengths: bilobalide (Rf about 0.75), ginkgolide A (Rf about 0.68), ginkgolide B (Rf about 0.52), ginkgolide J (Rf about 0.39), and ginkgolide C (Rf about 0.27). Other spots of varying intensities also may be seen.

Stems and other foreign organic matter

Not more than 3.0% of stems and not more than 2.0% of other foreign organic matter.

Loss on drying

Dry 1.0g of finely powdered Ginkgo Leaf at 105° for 2 hours: it loses not more than 11.0% of its weight. Reserve the dried test specimen for use in Identification test B.

Total ash

Not more than 11.0% determined on 1.0g of finely powdered Ginkgo Leaf.

Heavy metals

[To come]

Pesticide residues

[To come]

Microbial limits

The total bacterial count does not exceed 10,000 per g, the total combined molds and yeasts count does not exceed 100 per g, and it meets the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus.

Content of flavonol glycosides

Extraction solvent — Prepare a 60% (w/w) solution of acetone in water.

Hydrochloric acid solution — Transfer 10.0mL of hydrochloric acid to a 50-mL volumetric flask, dilute with water to volume, and mix.

Mobile phase — Prepare a mixture of citric acid solution (0.6 in 100), acetonitrile, and isopropyl alcohol (100:47:5). Make adjustments if necessary.

Standard solutions — Dissolve an accurately weighed quantity of USP Quercetin RS in methanol to obtain a solution having a known concentration of about 0.8mg per mL (Standard stock solution). To three separate 100-mL volumetric flasks, transfer 3.0mL, 5.0mL, and 10.0mL of Standard stock solution, and add 10mL of water followed by 30mL of Hydrochloric acid solution to each flask. Dilute the contents of each flask with methanol to volume, and mix to obtain Standard solutions A, B, and C having known concentrations of 0.024mg, 0.04mg, and 0.08mg per mL, respectively.

Test solution — Transfer about 2.5g of Ginkgo Leaf, finely powdered and accurately weighed, to a suitable flask fitted with a reflux condenser. Add 50mL of Extraction solvent, and heat on a hot water bath, under reflux, for 30 minutes. Allow to cool, filter, and collect the filtrate in a 100-mL volumetric flask. Extract the residue on the filter a second time in the same manner using 40mL of Extraction solvent, and collect the filtrate in the same 100-mL volumetric flask. Dilute the contents of the flask with Extraction solvent to volume, and mix. Evaporate 50.0mL of the solution to dryness, and transfer the residue to a 50-mL volumetric flask with the aid of 30mL of methanol. Add 4.4mL of Hydrochloric acid solution, dilute with water to volume, mix and centrifuge. Transfer 10.0mL of the supernatant liquid to a suitable amber-glass vial, close with a rubber seal and an aluminum cap, heat in a boiling water bath for 25 minutes and allow to cool to room temperature.

Chromatographic system — The liquid chromatograph is equipped with a 370-nm detector and a 4.6-mm х 12.5-cm column that contains packing LI. The flow rate is about 1.5mL per minute. Chromatograph Standard solution A, and record the peak responses as directed under Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure — Separately inject equal volumes (about 10µL) of each of the Standard solutions and the Test solution into the chromatograph, record the chromatograms, measure the areas of the responses for the major peaks, and add the peak areas of all the major peaks due to quercetin, kaempferol and isorhamnetin in the chromatogram of the Test solution. The relative retention times of the flavone glycosides of interest are about 1.0 for quercetin, 1.6 for kaempferol, and 1.7 for isorhamnetin. [Note — Isorhamnetin sometimes co-elutes with kaempferol]. Plot the peak areas of responses of the major peaks of the Standard solutions versus concentrations, in mg per mL, of USP Quercetin RS and draw the straight line best fitting the three plotted points. From the graph so obtained determine the concentration, in mg per mL, of flavone glycosides in the Test solution. Multiply the values obtained by a mean molecular mass factor of 2.514 and calculate the percentage of flavonol glycosides, as quercetin, with a mean molecular mass of 756.7: not less than 0.8% of flavone glycosides is found.

In 2010, a meta-analysis of clinical trials has shown Ginkgo to be moderately effective in improving cognition in dementia patients.