Macroscopic— It shows fragments of dark brown, lignified branches, usually from 1 mm to not more than 2.5 mm in diameter, bearing alternate petiolate leaves, with small, often deciduous styles, and bearing numerous white flowers in a corymbose arrangement. The leaves are more or less strongly lobate, and their margins are slightly or very slightly serrate. C. laevigata has pinnatilobate to pinnatifid leaves, divided into three, five, or seven obtuse lobes; the leaves of C. monogyna are almost pinnatisect with three to five acute lobes. The adaxial surface of the leaf is dark green to brownish green; the abaxial surface is lighter, greyish green, and shows a dense network of clearly visible veinlets and slightly prominent principal veins. The leaves of C. laevigata and C. monogyna are glabrous or bear isolated trichomes. The flowers consist of a brownish-green tubulous calyx, ending in its upper part in five triangular segments, and of five yellowish white to brownish free petals, rounded to widely oval, shortly unguiculate, and with numerous stamens. The ovary, fused to the tubulous calyx, bears one to three long styles and consists of the same number of carpels, each containing one fertile ovule. C. monogyna has one style and one carpel, and C. laevigata has two or three styles and carpels.

Microscopic— When reduced to a fine powder and examined under a microscope, the yellowish-green powder shows the following characteristics: unicellular covering trichomes, usually with thick walls and wide lumens, almost straight to somewhat curved, pitted at the base; fragments of leaf epidermis with cells that have sinuous to polygonal walls and large anomocytic stomata surrounded by four to seven subsidiary cells; clusters of parenchymatous cells containing calcium oxalate crystals, usually from 10 to 20 µm in length; fragments of petals showing rounded polygonal epidermal cells, strongly papillose, with thick walls, the cuticle of which clearly shows wavy striations; fragments of anthers whose endothecium has an arched and regularly thickened margin; fragments of stems containing collenchymatous cells, vessels and fibers of lignified sclerenchyma, with narrow lumens; numerous rounded to elliptical triangular pollen grains up to 45 µm in diameter, with free exines and three germinal pores.

A: Thin-Layer Chromatographic Identification Test 201—

B: Solution A—Prepare a filtered and degassed mixture of tetrahydrofuran, methanol, and acetonitrile, (92.4:4.2:3.4).

Solution A— Prepare a 0.5% solution of phosphoric acid in water.

Solution B— Prepare a mixture of tetrahydrofuran, isopropyl alcohol, and acetonitrile (10:8:3).

Mobile phase— Prepare a filtered and degassed mixture of Solution A and Solution B (88:12). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of USP Vitexin RS in Solution B, with heating if necessary, to obtain a solution having a known concentration of about 0.3 mg per mL.

Test solution— Weigh and finely powder about 100 g of Hawthorn Leaf with Flower. Transfer about 4 g of the powder, accurately weighed, to a continuous-extraction apparatus fitted with a flask containing about 80 mL of methanol, and extract the test specimen for 5 hours. Cool, remove the flask, and evaporate the solvent from the extract under vacuum to about 40 mL. Transfer this solution to a 50-mL volumetric flask, dilute with methanol to volume, and mix. Transfer 10.0 mL of the solution to a suitable flask fitted with a reflux condenser, add 4 mL of 25% hydrochloric acid, and heat the flask under reflux on a water bath at 65 for 90 minutes. Cool, transfer the contents of the flask to a 50-mL volumetric flask, dilute with methanol to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 336-nm detector and a 4-mm × 10-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the column efficiency is not less than 3000 theoretical plates; the tailing factor is between 0.8 and 2; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 5 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas of the major peaks. Calculate the percentage of C-glycosylated flavones, expressed as vitexin (C21H20O10), in the portion of Hawthorn Leaf with Flower taken by the formula: in which C is the concentration, in mg per mL, of USP Vitexin RS in the Standard solution; W is the weight, in g, of Hawthorn Leaf with Flower taken to prepare the Test solution; SrU is the sum of the peak areas of vitexin and isovitexin, with a relative retention time of about 1.0 and 0.85, respectively, in the chromatogram of the Test solution; and rS is the vitexin peak area obtained from the Standard solution.

Mobile phase— Prepare a mixture of methanol, water, and phosphoric acid (100:100:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of USP Quercetin RS in methanol, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 0.05 mg per mL.

Test solution— Proceed as directed for Test solution under Content of C-glycosylated flavones, except to use 1 mL of 25% hydrochloric acid for 60 minutes instead of 4 mL of 25% hydrochloric acid for 90 minutes.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 370-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the column efficiency is not less than 3000 theoretical plates; the tailing factor is between 0.8 and 2; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas of the major peaks. Calculate the percentage of O-glycosylated flavones, expressed as hyperoside (C21H20O12), in the portion of Hawthorn Leaf with Flower taken by the formula: in which 464.38 and 302.24 are the molecular weights of hyperoside and quercetin, respectively; C is the concentration, in mg per mL, of USP Quercetin RS in the Standard solution; and rU and rS are the quercetin peak areas in the chromatograms obtained from the Test solution and Standard solution, respectively.