Maritime Pine
» Maritime Pine consists of the bark of stems of Pinus pinaster Aiton (Pinus maritima Poir.) Fam. Pinaceae. It contains not less than 8.0 percent and not more than 12.0 percent 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 15 and 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 1 to 3 cm 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 50 or 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, 3 to 15 mm 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, 3 to 15 mm 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, 15 to 35 mm thick, separated by collapsed phellogen. Phelloderm and phellum cells are up to 100 mm 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 1 g of the dried Maritime Pine. Add 10 mg of the powdered material to 1 mL of methanol. Add 6 mL of a mixture of butanol and hydrochloric acid (95:5 v/v). Heat for 2 minutes in a water bath: the solution turns red.
B: Thin-Layer Chromatographic Identification Test 201
Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.
Test solution— Add 2 g of the powdered dried material to 20 mL of water. Place in a water bath for 20 minutes, and centrifuge. Extract the supernatant with 40 mL of ethyl acetate. Evaporate the ethyl acetate layer to dryness under a stream of nitrogen, with gentle heating. Dissolve the residue so obtained in 0.25 mL of alcohol.
Standard solution— Prepare a solution of USP Maritime Pine Extract RS in alcohol, having a concentration of about 25 mg per mL. [note—Retain a portion of this solution for use in Identification test C.]
Application volume: 5 µL.
Developing solvent system: a mixture of ethyl acetate, formic acid, 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 the plate with the Spray reagent, and heat at 115 for 15 minutes. Three red bands appear in the middle third of the chromatogram of the Standard solution corresponding to two dimeric procyanidins and catechin. The chromatogram of the Standard solution also exhibits a blue band between the upper band due to upper dimeric procyanidins and the band due to catechin. The chromatogram of the Test solution contains bands that correspond to those found in the chromatogram of the Standard solution.
C: Thin-Layer Chromatographic Identification Test 201
Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.
Test solution— Use the Test solution prepared as directed for Identification test B.
Standard solution 1— Use the Standard solution prepared as directed for Identification test B.
Standard solution 2— Prepare a solution of ferulic acid and protocatechuic acid containing 1 mg of each per mL.
Application volume: 10 µL.
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 110 and to examine the plate under short-wavelength and long-wavelength UV light. The chromatograms of Standard solution 1 and Standard solution 2 exhibit bands in the middle third and upper third that correspond to protocatechuic acid and ferulic acid, respectively. Spray the plate with the Spray reagent, and heat at 115 for 15 minutes. The bands due to ferulic acid and protocatechuic acid turn grayish green. Grayish-green bands become visible in the chromatogram of Standard solution 1 above and below protocatechuic acid, indicating the presence of caffeic acid and catechin, respectively. The chromatogram of the Test solution exhibits bands due to catechin, protocatechuic acid, caffeic acid, and ferulic acid that correspond in color and RF values to those in the chromatogram of Standard solution 1 and Standard solution 2.
Water content 561: not more than 35.0%.
Foreign organic matter 561: not more than 5%.
Total ash 561: 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 2 g of ferric ammonium sulfate in a mixture of 100 mL of water and 17.5 mL of hydrochloric acid. [note—This solution can be used within 15 days of preparation.]
Standard solution— Prepare a solution of USP Maritime Pine Extract RS in methanol having a concentration of about 95 µg of procyanidins per mL.
Test solution— Dry crushed Maritime Pine at 110 for 3 hours. Place about 1.9 g of the crushed material, accurately weighed, in a 20-mL vial, and add 10 mL of methanol. Crimp the vial, and sonicate for 2 minutes. Heat in boiling water for 10 minutes. Cool to room temperature, allow the sediment to settle, and transfer the supernatant to a 100-mL volumetric flask, passing it through a filter having a 0.45-µm porosity. Wash the sediment two times with 10 mL of methanol, and transfer the solution into the same 100-mL volumetric flask, again passing it through a filter having a 0.45-µm porosity. Dilute with methanol to volume, and mix. Transfer 1.0 mL of that solution into a 20-mL volumetric flask, dilute with methanol to volume, and mix.
Procedure— Transfer 1.0 mL of the Standard solution, 1.0 mL of the Test solution, and 1.0 mL of methanol to three separate 10-mL vials. To each vial add 6.0 mL of Reagent solution A and 0.25 mL of Reagent solution B. Seal the vials with crimp caps. Mix, and heat in a water bath for 40 minutes. Quickly cool to room temperature in an ice bath. Quantitatively transfer these solutions, with the aid of Reagent solution A, to three separate 10-mL volumetric flasks, dilute with Reagent solution A to volume, and mix. Determine the absorbance of the solutions obtained from the Standard solution and the Test solution at 551 nm, using the methanol-containing solution as the blank. Calculate the percentage of total procyanidins in the portion of Maritime Pine taken by the formula:
200(AU /AS)(CS /W)
in which AU and AS are the absorbances of the solutions from the Test solution and the Standard solution, respectively; CS is the concentration, in µg per mL, of the Standard solution; and W is the weight, in mg, of Maritime Pine powder used to prepare the Test solution, corrected for the water content.
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Maged H. Sharaf, Ph.D.
Senior Scientist
1-301-816-8318
(DSB05) Dietary Supplements - Botanicals
Reference Standards Lili Wang, Technical Services Scientist
1-301-816-8129
RSTech@usp.org
USP32–NF27 Page 1050
Pharmacopeial Forum: Volume No. 32(4) Page 1140