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.
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, 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 the
Spray reagent, and dry at 110
for 10 minutes. A red band appears in the upper part of the chromatogram of the
Test solution, at an
RF value of about 0.82, corresponding to a similar band in the chromatogram of the
Standard solution (presence of catechin). The lower part of the chromatogram of the
Test solution also shows red bands, at an
RF value of about 0.45
(presence of oligomeric and polymeric procyanidins). Two other red bands in the chromatogram of the
Test solution correspond to those at similar
RF values in the chromatogram of the
Standard solution (presence of dimeric procyanidins). A blue band appears in the chromatogram of the
Test solution between the bands for catechin and the dimeric procyanidins, corresponding in color and
RF value to a similar band 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 examine the plate under short-wavelength UV light. The upper third of the chromatogram of the
Test solution exhibits three bands. The uppermost band is close to the solvent front. The middle third of the chromatogram of the
Test solution exhibits a band corresponding in
RF value to the bands in the chromatograms of
Standard solution 1 and
Standard solution 2 (presence of ferulic acid). The lower third of the chromatogram of the
Test solution exhibits a band corresponding to bands of similar
RF value in the chromatograms of
Standard solution 1 and
Standard solution 2 (presence of protocatechuic acid). A band near the origin is also visible in the chromatogram of the
Test solution. Spray the plate with the
Spray reagent, and dry at 110
. The bands due to ferulic acid and protocatechuic acid turn grayish green and orange, respectively. A grayish-green band becomes visible in the chromatogram of the
Test solution above the protocatechuic acid band
(presence of caffeic acid). The band near the origin of the chromatogram of the
Test solution turns orange.
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.]
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
Test solution and 1.0 mL of methanol to two separate 10-mL vials. To each flask add 6.0 mL of
Reagent solution A and 0.25 mL of
Reagent solution B to each flask. 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 two separate 10-mL volumetric flasks, dilute with
Reagent solution A to volume, and mix. Determine the absorbance of the solution obtained from the
Test solution at 546 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:
(2000AU )/(36.7W),
in which
AU is the absorbance of the solution obtained from the
Test solution; 36.7 is the absorptivity of the maritime pine procyanidins; and
W is the weight, in g, of the Maritime Pine taken to prepare the
Test solution.
