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Maritime Pine Extract
» Maritime Pine Extract is prepared from the pulverized Maritime Pine using suitable solvents. It contains between 65 and 75 percent of procyanidins, calculated on the dried basis.
Packaging and storage— Preserve in tight containers, and store at 25, excursion permitted between 15 and 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 for Labeling under Botanical Extracts 565.
Identification—
A: Dissolve 50 mg of Extract in 6 mL of a mixture of butanol and hydrochloric acid (95:5). Heat in a water bath for 2 minutes: the solution turns dark red.
B: Thin-Layer Chromatographic Identification Test 201—
Test solution— Dissolve a quantity of Extract in methanol to obtain a solution having a concentration of about 25 mg per mL.
Standard solution 1— Prepare a solution of USP Maritime Pine Extract RS in methanol having a concentration of about 25 mg per mL.
Standard solution 2— Prepare a solution of ferulic acid in methanol having a concentration of about 1 mg per mL.
Application volume: 5 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 chromatogram of Standard solution 1 exhibits 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 dry at 110 for 10 minutes. The bands due to ferulic acid and protocatechuic acid turn grayish green and orange, respectively. 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 caffeic acid, protocatechuic acid, and ferulic acid that correspond in color and RF value to those in the chromatogram of Standard solution 1.
C: Thin-Layer Chromatographic Identification Test 201—
Test solution— Use the Test solution prepared as directed for Identification test B.
Standard solution— Use the Standard solution 1 prepared as directed for Identification test B.
Application volume: 5 µL.
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 the Spray reagent and heat at 110 for 10 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.
D: Proceed as directed in the following liquid-chromatographic procedure.
Solution A— Use filtered and degassed methanol.
Solution B— Carefully weigh 1 g of phosphoric acid, and dilute with water. Transfer to a 1000-mL volumetric flask, dilute with water to volume, and mix.
Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).
Standard solution— Dissolve an accurately weighed quantity of USP Maritime Pine Extract RS in Solution A to obtain a solution having a known concentration of about 2 mg per mL. Pass through a membrane having a 0.45-µm or finer porosity.
Test solution— Weigh about 20 mg of Extract. Add 10 mL of Solution A, and sonicate for 10 minutes. Pass through a membrane having a 0.45-µm or finer porosity, discarding the first 4 mL of the filtrate.
Chromatographic system (see Chromatography 621)— 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 1.0 mL per 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 10 µL) of the Standard solution and the Test solution into 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 the Standard solution with the Reference Chromatogram: the chromatogram of the Test solution exhibits peaks for catechin, caffeic acid, taxifolin, and ferulic acid at the retention times corresponding to those in the chromatogram of the Standard solution.
Microbial enumeration 2021— The total aerobic microbial count does not exceed 104 cfu per g, the total combined molds and yeasts count does not exceed 1000 cfu per g, and it meets the requirements of the tests for absence of Salmonella species, and Escherichia coli.
Loss on drying 731— Dry about 1.0 g of Extract, accurately weighed, for 3 hours at 110: it loses not more than 8.0% of its weight.
Total ash 561: not more than 0.7%.
Pesticide residue 561: meets the requirement.
Limit of water-insoluble substances— Accurately weigh 0.50 g of Extract, and stir in 50 mL of water at 20 for 15 minutes. Pass through a fine sintered glass filter, previously weighed. Dry the filter at 110 for 3 hours, 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 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— Transfer about 0.125 g of Extract, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with methanol to volume, and mix. Transfer 1.0 mL of this solution to 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. 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 separate10-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 Extract 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 Extract taken to prepare the Test solution.
Residual solvents 467: meet the requirements.
(Official January 1, 2007)
Other requirements— It meets the requirements for Heavy Metals under Botanical Extracts 565.
Auxiliary Information— Staff Liaison : Maged H. Sharaf, Ph.D., Senior Scientist
Expert Committee : (DSB05) Dietary Supplements - Botanicals
USP29–NF24 Page 2360
Pharmacopeial Forum : Volume No. 29(4) Page 1282
Phone Number : 1-301-816-8318