Maritime Pine Extract
»Maritime Pine Extract is prepared from the pulverized Maritime Pine using suitable solvents.It contains between 65and 75percent of procyanidins,calculated on the dried basis.
Packaging and storage— Preserve in tight containers,and store at 25,excursion permitted between 15and 30.Protect from light.
Labeling— The label states the official name of the article,the Latin binomial,and the part of the plant from which the article was prepared,in addition to the information required forLabelingunder Botanical Extracts á565ñ.
Identification—
A: Dissolve 50mg of Extract in 6mLof a mixture of butanol and hydrochloric acid (95:5).Heat in a water bath for 2minutes:the solution turns dark red.
B:Thin-Layer Chromatographic Identification Test á201ñ—
Test solution— Dissolve a quantity of Extract in methanol to obtain a solution having a concentration of about 25mg per mL.
Standard solution 1— Prepare a solution of USP Maritime Pine Extract RSin methanol having a concentration of about 25mg per mL.
Standard solution 2— Prepare a solution of ferulic acid in methanol having a concentration of about 1mg per mL.
Application volume: 5µL.
Developing solvent system: a mixture of methylene chloride,methanol,glacial acetic acid,and water (80:15:2:2).
Spray reagent— Prepare a 5%ferric chloride solution in methanol.
Procedure— Proceed as directed in the chapter,except to dry the plate at 110and to examine the plate under short-wavelength and long-wavelength UVlight.The chromatogram ofStandard solution 1exhibits bands in the middle third and upper third that correspond to protocatechuic acid and ferulic acid,respectively.Spray the plate with theSpray reagent,and dry at 110for 10minutes.The bands due to ferulic acid and protocatechuic acid turn grayish green and orange,respectively.Grayish-green bands become visible in the chromatogram ofStandard solution 1above and below protocatechuic acid indicating the presence of caffeic acid and catechin,respectively.The chromatogram of theTest solutionexhibits bands due to caffeic acid,protocatechuic acid,and ferulic acid that correspond in color andRFvalue to those in the chromatogram ofStandard solution 1.
C:Thin-Layer Chromatographic Identification Test á201ñ—
Test solution— Use theTest solutionprepared as directed forIdentificationtestB.
Standard solution— Use the Standard solution 1prepared as directed forIdentificationtestB.
Application volume: 5µL.
Developing solvent system: a mixture of ethyl acetate,formic acid,and water (100:10:6).
Spray reagent: a mixture of phosphoric acid and alcohol (1:1),containing 1%of vanillin.
Procedure— Proceed as directed in the chapter,except to spray the plate with theSpray reagent and heat at 110for 10minutes.Three red bands appear in the middle third of the chromatogram of theStandard solutioncorresponding to two dimeric procyanidins and catechin.The chromatogram of theStandard solutionalso exhibits a blue band between the upper band due to upper dimeric procyanidins and the band due to catechin.The chromatogram of theTest solutioncontains bands that correspond to those found in the chromatogram of theStandard solution.
D: Proceed as directed in the following liquid-chromatographic procedure.
Solution A— Use filtered and degassed methanol.
Solution B— Carefully weigh 1g of phosphoric acid,and dilute with water.Transfer to a 1000-mLvolumetric flask,dilute with water to volume,and mix.
Mobile phase— Use variable mixtures of Solution AandSolution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).
Standard solution— Dissolve an accurately weighed quantity of USP Maritime Pine Extract RSinSolution Ato obtain a solution having a known concentration of about 2mg per mL.Pass through a membrane having a 0.45-µm or finer porosity.
Test solution— Weigh about 20mg of Extract.Add 10mLof Solution A,and sonicate for 10minutes.Pass through a membrane having a 0.45-µm or finer porosity,discarding the first 4mLof the filtrate.
Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×15-cm column that contains base-deactivated packing L7,having less than 5-µm particle size.The column temperature is maintained at 40.The flow rate is about 1.0mLper minute.The chromatograph is programmed as follows.
Time
(minutes)		 Solution A
(%)		 Solution B
(%)		 Elution
0 8 92 equilibration
0–40 8®34 92®66 linear gradient
40–45 34®2 66®98 linear gradient
45–50 2 98 isocratic
50–52 2®8 98®92 linear gradient
52–57 8 92 isocratic
Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the chromatogram obtained is similar to the Reference Chromatogram provided with the USP Maritime Pine Extract RS;the resolution,R,between taxifolin and ferulic acid is not less than 3.0;and the tailing factor for taxifolin is not more than 2.0.
Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak areas for catechin,caffeic acid,taxifolin,and ferulic acid,identifying the peaks by comparison of the chromatogram of theStandard solution with the Reference Chromatogram:the chromatogram of theTest solutionexhibits peaks for catechin,caffeic acid,taxifolin,and ferulic acid at the retention times corresponding to those in the chromatogram of theStandard solution.
Microbial enumeration á2021ñ— The total aerobic microbial count does not exceed 104cfu per g,the total combined molds and yeasts count does not exceed 1000cfu per g,and it meets the requirements of the tests for absence of Salmonellaspecies,and Escherichia coli.
Loss on drying á731ñ— Dry about 1.0g of Extract,accurately weighed,for 3hours at 110:it loses not more than 8.0%of its weight.
Total ash á561ñ: not more than 0.7%.
Pesticide residue á561ñ: meets the requirement.
Limit of water-insoluble substances— Accurately weigh 0.50g of Extract,and stir in 50mLof water at 20for 15minutes.Pass through a fine sintered glass filter,previously weighed.Dry the filter at 110for 3hours,cool to room temperature,and weigh the filter.Calculate the amount of water-insoluble material:not more than 10%of the amount of Extract taken.
Content of procyanidins—
Reagent solution A— Prepare a mixture of butanol and hydrochloric acid (95:5).[NOTE—Prepare this solution on the day of use.]
Reagent solution B— Dissolve 2g of ferric ammonium sulfate in a mixture of 100mLof water and 17.5mLof hydrochloric acid.[NOTE—This solution can be used within 15days of preparation.]
Test solution— Transfer about 0.125g of Extract,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with methanol to volume,and mix.Transfer 1.0mLof this solution to a 20-mLvolumetric flask,dilute with methanol to volume,and mix.
Procedure— Transfer 1.0mLof theTest solutionand 1.0mLof methanol to two separate 10-mLvials.To each flask add 6.0mLofReagent solution Aand 0.25mLofReagent solution B.Seal the vials with crimp caps.Mix,and heat in a water bath for 40minutes.Quickly cool to room temperature in an ice bath.Quantitatively transfer these solutions,with the aid ofReagent solution A,to two separate10-mLvolumetric flasks,dilute withReagent solution Ato volume,and mix.Determine the absorbance of the solution obtained from theTest solutionat 546nm,using the methanol-containing solution as the blank.Calculate the percentage of total procyanidins in the portion of Extract taken by the formula:
(2000AU)/(36.7W),
in whichAUis the absorbance of the solution obtained from theTest solution;36.7is the absorptivity of the maritime pine procyanidins;andWis the weight,in g,of the Extract taken to prepare theTest solution.
Other requirements— It meets the requirements forHeavy MetalsunderBotanical Extracts á565ñ.
Auxiliary Information— Staff Liaison:Gabriel I.Giancaspro,Ph.D.,Senior Scientist and Latin American Specialist
Expert Committee:(DSB)Dietary Supplements:Botanicals
USP28–NF23Page 2115
Pharmacopeial Forum:Volume No.29(4)Page 1282
Phone Number:1-301-816-8343