Macroscopic— Horse chestnut seeds are dense and hard,subspherical to oval,slightly flattened,and from 2to 4cm in diameter.They have a dark brown seed coat from 1to 1.5mm thick,with a large,round,light brown spot (hilum).The seed coat is shiny,but only in fresh condition.The space under the coat is totally filled with the shiny,massive embryo and its large,pale yellow cotyledons lacking endosperm.

Microscopic— The epidermis of the testa in surface view has yellowish-brown cells of fairly uniform size,with the majority of cells rounded to polygonal,and a few that are square to obscurely triangular.The walls of these cells are considerably but rather unevenly thickened,and lack pits.In sectional view,the cells are columnar,approximately 3to 4times as high as they are wide,with the outer periclinal wall markedly thickened,uneven,and becoming thinner towards the base;beneath the epidermis there are a few layers of small collenchymatously thickened cells with small intercellular spaces;the greater part of the testa consists of larger,loosely-packed parenchymatous cells forming a spongy tissue;the walls are variably and unevenly thickened,with intracellular and large circular spaces well marked;the inner testa is a narrow zone,with ill-defined and thinner-walled cells.All the parenchymatous cells of the testa are darkly pigmented.The embryo has an outer layer of small colorless cells,almost square in sectional view,with outer and side walls thickened.In surface view,only the irregular and more or less polygonal lumens are discernible,giving a reticulate,pitted appearance.Cotyledons are moderately thickened and indistinctly pitted,having round to ovoid parenchymatous cells densely filled with starch.Starch granules,mainly simple,are present in two size ranges:from 15to 30µm and from 3to 10µm.The largest granules vary from circular,ovoid,and bluntly polygonal to pyriform,most of them with a well-marked cleft or stellate hilum,and lacking striations.The smaller starch granules are less variable,spherical to ovoid,with the hilum more often a point.Compound starch granules are very infrequently found.

Test solution— Transfer about 1g of the powdered plant material to a screw-capped centrifuge tube,add 10mLof a mixture of alcohol and water (7:3),and heat on a steam bath for 10minutes.Centrifuge,and use the clear supernatant.

Standard solution— Dissolve an accurately weighed quantity of USP Escin RSin methanol to obtain a solution having a known concentration of about 5mg per mL.

Developing solvent system— Use the upper phase of a mixture of 1-butanol,water,and glacial acetic acid (50:40:10).

Spray reagent— Prepare a mixture of methanol,glacial acetic acid,sulfuric acid,and p-anisaldehyde (85:10:5:0.5).

Procedure— Develop the chromatogram to a length of not less than 15cm,and dry the plate in a current of air.Spray the plate with Spray reagent,heat the plate at 100for 5minutes,and examine the plate under daylight:the chromatogram obtained from the Test solutionshows a blue-violet zone corresponding to escin,comparable in position and color to the main zone in the chromatogram obtained from the Standard solution.Above this zone,the chromatogram of the Test solutionshows several narrow,brown to brownish-red zones that are less intense than the zone corresponding to escin.

Change to read:

Solvent 1— Prepare a mixture of methanol and water (65:35).

Solvent 2— Use the lower phase of a mixture of 30mLof 0.1Nhydrochloric acid,20mLof 1-propanol,and 50mLof chloroform.

Reagent— Dissolve 75mg of ferric chloride in 50mLof glacial acetic acid.Add 50mLof sulfuric acid,while shaking and cooling.Prepare immediately before use.

Escin standard solutions— Dissolve an accurately weighed quantity of USP Escin RSin glacial acetic acid,shaking for 1minute.Dilute quantitatively,and stepwise if necessary,to obtain solutions having known concentrations of about 0.6,0.4,and 0.2mg per mL.

Test solution— Accurately weigh 1.00g of ground seeds,and place in a 250-mLround-bottom flask.Add exactly 100mLof Solvent 1,and weigh the filled flask with a precision of ±0.1g.Attach a condenser to the flask,reflux for 30minutes,and allow to cool.Adjust to the initial weight by adding Solvent 1as needed,mix,and filter.Transfer 30.0mLof the filtrate to a round-bottom flask,and evaporate the solvents under vacuum.Dissolve the residue with 20mLof 0.1Nhydrochloric acid,and quantitatively transfer with the aid of two additional 5-mLportions of 0.1Nhydrochloric acid to a 250-mLseparation funnel.Add 20mLof 1-propanol and 50mLof chloroform,and shake vigorously for 2minutes.Separate the chloroform layer,and add Solvent 2to the upper phase remaining in the separation funnel.Shake vigorously for 2minutes,and separate the chloroform layer.Combine the chloroform layers in a round-bottom flask,and evaporate to dryness under vacuum.Evaporate the remaining solvents with the aid of a current of air.Wash the residue with two 10-mLportions of ether,filter,wash the filter with 10mLof ether,and discard the ether filtrates.After evaporation of the residual ether,add to the residue a 10-mLportion of glacial acetic acid,and pass through the previously used dried filter into a 50-mLvolumetric flask.Repeat the addition of glacial acetic acid followed by filtration two additional times,combining the filtrates in the volumetric flask.Wash the round-bottom flask with small quantities of glacial acetic acid,and filter into the volumetric flask.Dilute with glacial acetic acid to volume.

Procedure— Transfer 1mLeach of theEscin standard solutions,the Test solution,and glacial acetic acid to separate test tubes with stoppers.Add 4.0mLof Reagentto each tube,cap the tubes,and place them in a water bath at 60for 25minutes,shaking occasionally.Measure the absorbances at 540nm of the reacted Test solutionand the reacted Escin standard solutions,using glacial acetic acid as the blank.Plot the absorbances obtained from the reacted Escin standard solutionsversus concentrations,in mg per mL,of USP Escin RSin the corresponding Escin standard solution.From the graphs so obtained,determine the concentration,C,in mg per mL,of triterpene glycosides as escin (C55H86O24)in the Test solution.Calculate the percentage of triterpene glycosides in the portion of Horse Chestnut taken by the formula: in which Cis the concentration,in mg per mL,of triterpene glycosides in the Test solutionas obtained above;and Wis the weight,in g,of Horse Chestnut taken to prepare the Test solution.