Maritime Pine
»Maritime Pine consists of the bark of stems ofPinus pinaster Aiton (Pinus maritima Poir.)Fam.Pinaceae.It contains not less than 8.0percent and not more than 12.0percent of procyanidins,calculated on the dried basis.
NOTE—This article is intended to be used in the preparation of extracts only and is not for direct human consumption.
Packaging and storage—
Store at 25
,excursion permitted between 15and 30.Preserve in a well-closed container,and protect from moisture and excessive heat.
,excursion permitted between 15and 30.Preserve in a well-closed container,and protect from moisture and excessive heat.
Labeling—
The label states the Latin binomial and,following the official name,the part of the plant contained in the article.
Botanical characteristics—
Macroscopic—
Bark pieces are typically 1to 3cm thick.The inner bark is plane to slightly concave,whitish to light brown,striped longitudinally;shiny and of slightly irregular surface,only a few millimeters thick.Abrupt change to a sequence of hard,convex,nearly parallel layers alternating with smooth,light brown layers.Up to 50or more layers present,depending on the age of the bark.Outer surface of bark is dark reddish brown composed of irregular scaly patches,with deep V-shaped fissures.Outer surface may also be gray,gray-green,or green-yellow due to presence of lichens.
Microscopic (transverse section of bark)—
Light inner bark has irregular lateral stripes consisting of three to five cell layers of long,slender sieve cells with large pitted horizontal cell walls and large polygonal parenchyma cells containing single,irregular,rounded starch grain,3to 15mm wide.Lateral stripes are separated from each other by ray parenchyma cells.Ray parenchyma cells are homogeneous in appearance,one to four cell layers thick and four and twenty cell layers high,each cell containing single,irregular,rounded starch grain,3to 15mm wide.Cylindrical parenchyma cells with thin cell wall arranged in vertical rows with calcium oxalate prisms are also present.Outer part of the inner bark contains plate-shaped cells of undifferentiated periderm and older periderm with multiple layers of phellogen.The phellogen grows three to seven rows of phellum to the exterior and two to four rows of small cell phelloderm to the interior.The oldest and outermost part of the bark is composed of lignified sections of phelloderm and phellum cells,15to 35mm thick,separated by collapsed phellogen.Phelloderm and phellum cells are up to 100mm wide,square,rectangular,polygonal,or irregularly shaped.The cell walls are colorless.Phelloderm cells are moderately pitted with a reddish-brown content.Phellum cells have a thicker cell wall,strongly pitted,undulated contour,and a yellowish-brown to brownish-red content.Radially in between layers of phelloderm and phellum are layers of ray parenchyma cells,five to eight cells thick,rounded to radially stretched,thin walled,strongly pitted with collapsed cells and dead sieve cells.
Identification—
A:
Pulverize 1g of the dried Maritime Pine.Add 10mg of the powdered material to 1mLof methanol.Add 6mLof a mixture of butanol and hydrochloric acid (95:5v/v).Heat for 2minutes in a water bath:the solution turns red.
B:Thin-Layer Chromatographic Identification Test á201ñ—
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture.
Test solution—
Add 2g of the powdered dried material to 20mLof water.Place in a water bath for 20minutes,and centrifuge.Extract the supernatant with 40mLof ethyl acetate.Evaporate the ethyl acetate layer to dryness under a stream of nitrogen,with gentle heating.Dissolve the residue so obtained in 0.25mLof alcohol.
Standard solution—
Prepare a solution of USP Maritime Pine Extract RSin alcohol,having a concentration of about 25mg per mL.[NOTE—Retain a portion of this solution for use inIdentificationtestC.]
Application volume:
5µL.
Developing solvent system:
a mixture of ethyl acetate,methanol,and water (100:10:6).
Spray reagent:
a mixture of alcohol and phosphoric acid (1:1),containing 1%of vanillin.
Procedure—
Proceed as directed in the chapter,except to dry the plate with the aid of a current of air,spray with theSpray reagent,and dry at 110for 10minutes.Ared band appears in the upper part of the chromatogram of theTest solution,at anRFvalue of about 0.82,corresponding to a similar band in the chromatogram of theStandard solution (presence of catechin).The lower part of the chromatogram of theTest solutionalso shows red bands,at anRFvalue of about 0.45(presence of oligomeric and polymeric procyanidins).Two other red bands in the chromatogram of theTest solutioncorrespond to those at similarRFvalues in the chromatogram of theStandard solution (presence of dimeric procyanidins).Ablue band appears in the chromatogram of theTest solution between the bands for catechin and the dimeric procyanidins,corresponding in color andRFvalue to a similar band in the chromatogram of theStandard solution.
for 10minutes.Ared band appears in the upper part of the chromatogram of theTest solution,at anRFvalue of about 0.82,corresponding to a similar band in the chromatogram of theStandard solution (presence of catechin).The lower part of the chromatogram of theTest solutionalso shows red bands,at anRFvalue of about 0.45(presence of oligomeric and polymeric procyanidins).Two other red bands in the chromatogram of theTest solutioncorrespond to those at similarRFvalues in the chromatogram of theStandard solution (presence of dimeric procyanidins).Ablue band appears in the chromatogram of theTest solution between the bands for catechin and the dimeric procyanidins,corresponding in color andRFvalue to a similar band in the chromatogram of theStandard solution.
C:Thin-Layer Chromatographic Identification Test á201ñ—
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture.
Test solution—
Use theTest solution prepared as directed forIdentificationtestB.
Standard solution 1—
Use theStandard solution prepared as directed forIdentification testB.
Standard solution 2—
Prepare a solution of ferulic acid and protocatechuic acid containing 1mg of each per mL.
Application volume:
10µL.
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 examine the plate under short-wavelength UVlight.The upper third of the chromatogram of theTest solutionexhibits three bands.The uppermost band is close to the solvent front.The middle third of the chromatogram of theTest solutionexhibits a band corresponding inRFvalue to the bands in the chromatograms ofStandard solution 1andStandard solution 2(presence of ferulic acid).The lower third of the chromatogram of theTest solutionexhibits a band corresponding to bands of similarRFvalue in the chromatograms ofStandard solution 1andStandard solution 2(presence of protocatechuic acid).Aband near the origin is also visible in the chromatogram of theTest solution.Spray the plate with theSpray reagent,and dry at 110.The bands due to ferulic acid and protocatechuic acid turn grayish green and orange,respectively.Agrayish-green band becomes visible in the chromatogram of theTest solution above the protocatechuic acid band(presence of caffeic acid).The band near the origin of the chromatogram of theTest solution turns orange.
and examine the plate under short-wavelength UVlight.The upper third of the chromatogram of theTest solutionexhibits three bands.The uppermost band is close to the solvent front.The middle third of the chromatogram of theTest solutionexhibits a band corresponding inRFvalue to the bands in the chromatograms ofStandard solution 1andStandard solution 2(presence of ferulic acid).The lower third of the chromatogram of theTest solutionexhibits a band corresponding to bands of similarRFvalue in the chromatograms ofStandard solution 1andStandard solution 2(presence of protocatechuic acid).Aband near the origin is also visible in the chromatogram of theTest solution.Spray the plate with theSpray reagent,and dry at 110.The bands due to ferulic acid and protocatechuic acid turn grayish green and orange,respectively.Agrayish-green band becomes visible in the chromatogram of theTest solution above the protocatechuic acid band(presence of caffeic acid).The band near the origin of the chromatogram of theTest solution turns orange.
Water content á561ñ:
not more than 35.0%.
Foreign organic matter á561ñ:
not more than 5%.
Total ash á561ñ:
not more than 1.5%.
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—
Dry crushed Maritime Pine at 110for 3hours.Place about 1.9g of the crushed material,accurately weighed,in a 20-mLvial,and add 10mLof methanol.Crimp the vial,and sonicate for 2minutes.Heat in boiling water for 10minutes.Cool to room temperature,allow the sediment to settle,and transfer the supernatant to a 100-mLvolumetric flask,passing it through a filter having a 0.45-µm porosity.Wash the sediment two times with 10mLof methanol,and transfer the solution into the same 100-mLvolumetric flask,again passing it through a filter having a 0.45-µm porosity.Dilute with methanol to volume,and mix.Transfer 1.0mLof that solution into a 20-mLvolumetric flask,dilute with methanol to volume,and mix.
for 3hours.Place about 1.9g of the crushed material,accurately weighed,in a 20-mLvial,and add 10mLof methanol.Crimp the vial,and sonicate for 2minutes.Heat in boiling water for 10minutes.Cool to room temperature,allow the sediment to settle,and transfer the supernatant to a 100-mLvolumetric flask,passing it through a filter having a 0.45-µm porosity.Wash the sediment two times with 10mLof methanol,and transfer the solution into the same 100-mLvolumetric flask,again passing it through a filter having a 0.45-µm porosity.Dilute with methanol to volume,and mix.Transfer 1.0mLof that solution into a 20-mLvolumetric flask,dilute with methanol to volume,and mix.
Procedure—
Transfer 1.0mLof the Test solutionand 1.0mLof methanol to two separate 10-mLvials.To each flask add 6.0mLof Reagent solution Aand 0.25mLof Reagent solution Bto each flask.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 of Reagent solution A,to two separate 10-mLvolumetric flasks,dilute with Reagent solution Ato volume,and mix.Determine the absorbance of the solution obtained from the Test solutionat 546nm,using the methanol-containing solution as the blank.Calculate the percentage of total procyanidins in the portion of Maritime Pine taken by the formula:
(2000AU)/(36.7W),
in which AUis the absorbance of the solution obtained from theTest solution;36.7is the absorptivity of the maritime pine procyanidins;and Wis the weight,in g,of the Maritime Pine taken to prepare the Test solution.
Auxiliary Information—
Staff Liaison:Gabriel I.Giancaspro,Ph.D.,Senior Scientist and Latin American Specialist
Expert Committee:(DSB)Dietary Supplements:Botanicals
USP28–NF23Page 2114
Pharmacopeial Forum:Volume No.29(4)Page 1279
Phone Number:1-301-816-8343