Macroscopic— The rhizome is irregularly bent,about 3to 10mm thick,and light gray-brown on the outside;thin roots spring from the knotty bulges of a lengthwise furrow.Atransverse cut of the rhizome shows it is fibrous,light yellowish white,and usually has a small medulla cave.The roots are often very long,usually 0.5to 2mm thick,light yellow-brown on the outside,and contain some deep longitudinal furrows;a transverse cut shows a pale and almost pure-white color.

Histology— The transverse section of the rhizome and root shows the following characteristics.The rhizome has a narrow cork composed of brown,thin-walled cells,a few rows of tangentially elongated cortical parenchyma,and a pericyclic region with numerous fibers occurring singly or,more frequently,in small groups.Fibers are much elongated with very thick and lignified walls.Some cells of the pericycle and outer part of secondary phloem contain large globular compound crystals of calcium oxalate.The vascular cambial region is distinct and continuous with narrow radial groups of vascular tissue separated by wide medullary rays.The secondary phloem is mainly parenchymatous with groups of thin-walled sieve tissue.The xylem is dense and completely lignified,containing scattered vessels,isolated or in small groups,associated with moderately thickened xylem parenchyma cells and numerous thicker-walled xylem fibers with slit-shaped pits.Individual vessels have fairly large,closely arranged,bordered pits,while the adjacent parenchyma has simple or bordered pits.Medullary rays indicate alternating areas of lignified and unlignified cells,appearing as tangential bands between the vascular bundles,each composed of five or six layers of cells;the lignified cells have moderately thickened walls with simple pits.The pith is composed of rounded,unlignified parenchyma,collapsed in the central part to form a cavity.Mature roots show a thin cork,narrow phelloderm,and secondary phloem and xylem with alternating areas of lignified and unlignified parenchyma in the wide medullary rays,similar to that found in the rhizome.

Adsorbent: 0.50-mm layer of chromatographic silica gel mixture.

Test solution— Extract 1g of powder by refluxing with 10mLof a solution containing toluene,ethyl acetate,and methanol (7:2:1)for 15minutes,cool,and filter.Evaporate the filtrate to dryness under reduced pressure at less than 40,and dissolve the residue in 2mLof the toluene,ethyl acetate,methanol solution.

Standard solution— Dissolve an accurately weighed quantity of USP Scopoletin RSand USPb-Sitosterol RSin methanol to obtain a solution having a known concentration of 0.05and 0.5mg per mL,respectively.

Application volume: 20µLfor the Test solution;10µLfor the Standard solution.

Developing solvent system: diethyl ether and methanol (9:1).

Procedure— Proceed as directed in the chapter.Examine the plates under UVlight at 365nm.Spray the plate with about 10mLof a mixture of water,85%phosphoric acid,and 10%vanillin in 96%ethanol (4.5:4.5:1);heat between 100and 105for 10minutes;and examine under daylight.The chromatogram of the Test solutionexhibits a violet-red zone corresponding to the b-sitosterol peak at the same RFvalue as the b-sitosterol peak in the chromatogram of the Standard solution.Weakly violet-red zones above and below b-sitosterol,corresponding to b-sitosterol-glucoside,are visible.

pH5.5Acetate buffer— Mix 5.40g of anhydrous sodium acetate,0.3mLof glacial acetic acid,and water to a final volume of 100mL.

Reagent solution— Prepare a solution containing about 1.00g of ninhydrin,1.50g of hydrindantin,and 37.5mLof propylene glycol,and adjust with pH5.5Acetate bufferto 50.0mL.[NOTE—Prepare the Reagent solutiondaily.]

Standard solution— Dissolve accurately weighed quantities of USP Glutamic Acid RSand USP Aspartic Acid RSin water to obtain a solution having a known concentration of about 0.1mg of each per mL.

Test solution— Finely powder an amount of Stinging Nettle,and transfer about 1.0g,accurately weighed,to 80mLof water.Place in an ultrasonic bath for 25minutes,and centrifuge.Transfer the supernatant to a 100-mLvolumetric flask,dilute with water to volume,and filter.

Procedure— Transfer 5.0mLof the Test solutionand 1.0mLof the Standard solutionto two separate,appropriately labeled,50-mLvolumetric flasks.Add 4.0mLof water to the Standard solutionand 5.0mLof Reagent solutionto both the Test solutionand the Standard solution.Heat in a boiling water bath for 30minutes,cool,and adjust with a mixture of ethanol and water (1:1)to volume.Concomitantly determine the absorbances of the Standard solutionand the Test solutionin 1-cm cells at the wavelength of maximum absorbance at about 570nm with a suitable spectrophotometer.Prepare a blank using 5.0mLof water,and treat similarly to the Test solution.Calculate the percentage of total amino acids in the portion of Stinging Nettle taken by the formula: in which AUand ASare the absorbances of the Test solutionand the Standard solution,respectively;WSis the sum of the weights,in mg,of USP Glutamic Acid RSand USP Aspartic Acid RS,calculated on the dried basis,in the Standard solution;and WUis the weight,in mg,of dried Stinging Nettle in the Test solution:not less than 0.8%of total amino acids is found.

Derivatizing reagent— Prepare a solution containing equal volumes (1:1:1)of BSTFA[N,O-bis(trimethylsilyl)trifluoroacetamide],anhydrous pyridine,and a mixture of BSA[N,O-(trimethylsilyl)acetamide],TMSI(N-trimethylsilyimidazole),and TMCS(trimethylchlorosilane)(3:3:2).

Internal standard solution— Dissolve an accurately weighed quantity of cholesterol in chloroform to obtain a solution having a known concentration of about 10mg per mL.

Standard solution— Dissolve 50mg of USPb-Sitosterol RS,accurately weighed,in 2.0mLof chloroform,add 1mLof Internal standard solution,and dilute with chloroform to 5mL.Transfer 0.5mLof this solution to a 10-mLround-bottomed flask,dry the solvent under reduced pressure,add 1mLof Derivatizing reagent,and mix.

Test solution— Finely powder an amount of Stinging Nettle,transfer 50.0g to a Soxhlet apparatus,treat with chloroform,and extract for 6hours.The volume of chloroform used is at least twice the volume of the thimble with an appropriate-size flask.Dry the solvent under reduced pressure,add 1.0mLof Internal standard solution,and dilute with chloroform to 10mL.Transfer 0.5mLof this solution to a 10-mLround-bottomed flask,dry the solvent under reduced pressure,and add 0.5mLof Derivatizing reagent.

Chromatographic system (see Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector and a 0.20-mm ×25-m fused-silica capillary column coated with a 0.35-µm film of phase G2.The carrier gas is helium,flowing at a rate of about 0.5mLper minute.The injection port and detector temperatures are maintained at 325.The column temperature is initially held at 300and maintained at this temperature for 60minutes.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the tailing factor for each sterol peak is not more than 2.0;and the relative standard deviation for replicate injections determined from each sterol peak is not more than 5.0%.

Procedure— Separately inject equal volumes (about 1µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses of the sterols.Calculate the percentage of b-sitosterol in the portion of Stinging Nettle taken by the formula: in which RUand RSare the peak response ratios of b-sitosterol to the internal standard obtained from the Standard solutionand the Test solution,respectively;CSis the concentration,in mg per mL,of USPb-Sitosterol RSin the Standard solution;and CUis the concentration,in mg per mL,of Stinging Nettle in the Test solution:not less than 0.05%of b-sitosterol is found.

Solution A— Use water.

Solution B— Use methanol.

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution— Dissolve an accurately weighed quantity of USP Scopoletin RSin methanol to obtain a solution having a known concentration of about 0.02µg per mL.

Test solution— Finely powder an amount of Stinging Nettle,and mix 4.000g,accurately weighed,with 25mLof methanol.Place in an ultrasonic bath for 25minutes,and centrifuge.Transfer 0.5mLof the solution to a 10-mLvolumetric flask,and dilute with methanol to volume.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a fluorescence detector set at an excitation wavelength of 366nm and an emission wavelength of 420nm and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1mLper minute.The chromatograph is programmed as follows. Chromatograph about 10µLof the Standard solution,and record the peak responses as directed for Procedure:the capacity factor(k ¢)determined from the scopoletin peak is not less than 5;the tailing factor for the scopoletin peak is not more than 2.0;and the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses for scopoletin.Calculate the content of scopoletin (C10H8O4),in µg per g,in the portion of Stinging Nettle taken by the formula: in which rUand rSare the peak responses of scopoletin in the Test solutionand the Standard solution,respectively;CSis the concentration,in mg per mL,of USP Scopoletin RSin the Standard solution;and CUis the concentration,in mg per mL,of Stinging Nettle in the Test solution:not less than 3µg per g of scopoletin is found.