Maritime Pine Extract
DEFINITION
Maritime Pine Extract is prepared from the pulverized Maritime Pine using suitable solvents. It contains NLT 65% and NMT 75% of procyanidins, calculated on the dried basis.
IDENTIFICATION
• A. Presence of Procyanidins
Sample solution:
Dissolve 50 mg of Extract in 6 mL of a mixture of butanol and hydrochloric acid (95:5).
Analysis:
Heat for 2 min in a water bath.
Acceptance criteria:
The solution turns red.
• B. Thin-Layer Chromatographic Identification Test
Standard solution A:
25 mg/mL of USP Maritime Pine Extract RS in methanol
Standard solution B:
1 mg/mL each of ferulic acid and protocatechuic acid in methanol
Sample solution:
25 mg/mL of Extract in methanol
Chromatographic system
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture
Application volume:
5 µL
Developing solvent system:
Methylene chloride, methanol, glacial acetic acid, and water (80:15:2:2)
Spray reagent:
5% ferric chloride solution in methanol
Analysis
Samples:
Standard solution A, Standard solution B, and Sample solution
Develop the chromatogram, dry the plate at 110
, and examine the plate under short-wavelength and long-wavelength UV light. The chromatograms of Standard solution A and Standard solution B 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 min. The bands due to ferulic acid and protocatechuic acid turn grayish green. Grayish-green bands become visible in the chromatogram of Standard solution A above and below protocatechuic acid, indicating the presence of caffeic acid and catechin, respectively.
, and examine the plate under short-wavelength and long-wavelength UV light. The chromatograms of Standard solution A and Standard solution B 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 min. The bands due to ferulic acid and protocatechuic acid turn grayish green. Grayish-green bands become visible in the chromatogram of Standard solution A above and below protocatechuic acid, indicating the presence of caffeic acid and catechin, respectively.
Acceptance criteria:
The chromatogram of the Sample 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 A and Standard solution B.
• C. Thin-Layer Chromatographic Identification Test
Standard solution:
Use Standard solution A, prepared as directed for Identification test B.
Sample solution:
Use the Sample solution, prepared as directed for Identification test B.
Chromatographic system
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture
Application volume:
5 µL
Developing solvent system:
Ethyl acetate, formic acid, and water (50:5:3)
Spray reagent:
Phosphoric acid and alcohol (1:1), containing 1% of vanillin
Analysis
Samples:
Standard solution and Sample solution
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 min. 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.
for 15 min. 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.
Acceptance criteria:
The chromatogram of the Sample solution contains bands that correspond to those found in the chromatogram of the Standard solution.
• D. HPLC Identification Test
Solution A:
Methanol
Solution B:
1 mg/mL of phosphoric acid in water
Mobile phase:
See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 8 | 92 |
| 40 | 34 | 66 |
| 45 | 2 | 98 |
| 50 | 2 | 98 |
| 52 | 8 | 92 |
| 57 | 8 | 92 |
Standard solution:
2 mg/mL of USP Maritime Pine Extract RS in Solution A. Pass through a membrane having a 0.45-µm or finer pore size.
Sample solution:
Add 20 mg of Extract to 10 mL of Solution A, and sonicate for 10 min to dissolve. Pass through a membrane having a 0.45-µm or finer pore size, discarding the first 4 mL of the filtrate.
Chromatographic system
Mode:
LC
Detector:
UV 280 nm
Column:
4.6-mm × 15-cm; base-deactivated packing L7, having less than 5-µm particle size
Column temperature:
40
Flow rate:
1 mL/min
Injection size:
10 µL
System suitability
Sample:
Standard solution
Suitability requirements
Chromatogram similarity:
The chromatogram is similar to the reference chromatogram provided with the lot of USP Maritime Pine Extract RS being used.
Resolution:
NLT 3.0 between taxifolin and ferulic acid
Tailing factor:
NMT 2.0 for taxifolin
Analysis
Samples:
Standard solution and Sample solution
Identify the peaks for catechin, caffeic acid, taxifolin, and ferulic acid by comparison of the chromatogram of the Standard solution with the reference chromatogram.
Acceptance criteria:
The chromatogram of the Sample 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.
COMPOSITION
• Content of Procyanidins
Reagent solution A:
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:
95 µg/mL of procyanidins from USP Maritime Pine Extract RS in methanol
Sample stock solution:
Transfer 250 mg of Extract to a 100-mL volumetric flask. Dissolve with methanol, and dilute with the same solvent to volume.
Sample solution:
Dilute the Sample stock solution with methanol (1 in 20).
Analysis
Samples:
Standard solution and Sample solution
Transfer 1.0 mL each of the Standard solution, Sample solution, and 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 min. 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, and dilute with Reagent solution A to volume.
Determine the absorbance of the solutions obtained from the Standard solution and the Sample solution, using the methanol-containing solution as a blank.
Calculate the percentage of total procyanidins in the portion of Extract taken:
Result = (AU/AS) × CS × (V/W) × D × P
| AU | = | = absorbance of the solution from the Sample solution |
| AS | = | = absorbance of the solution from the Standard solution |
| CS | = | = concentration of the USP Maritime Pine Extract RS in the Standard solution (mg/mL) |
| V | = | = volume of the Sample stock solution (mL) |
| W | = | = weight of Maritime Pine Extract taken to prepare the Sample stock solution (mg) |
| D | = | = dilution factor to prepare the Sample solution from Sample stock solution, 20 |
| P | = | = percentage of procyanidines in the USP Maritime Pine Extract RS |
Acceptance criteria:
65%–75% on the dried basis
CONTAMINANTS
• Botanical Extracts, Heavy Metals 
565
:
Meets the requirements
565:
Meets the requirements
• Articles of Botanical Origin, Pesticide Residue 561:
Meets the requirements
561:
Meets the requirements
• Microbial Enumeration Tests 2021:
The total aerobic microbial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.
2021:
The total aerobic microbial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.
• Absence of Specified Microorganisms 2022:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
2022:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
SPECIFIC TESTS
• Loss on Drying 731:
Dry 1.0 g of Extract for 3 h at 110: it loses NMT 8.0% of its weight.
731:
Dry 1.0 g of Extract for 3 h at 110: it loses NMT 8.0% of its weight.
• Articles of Botanical Origin, Total Ash 561:
NMT 0.7%
561:
NMT 0.7%
• Limit of Water-Insoluble Substances
Analysis:
Weigh 0.50 g of Extract, and stir in 50 mL of water at 20 for 15 min. Pass through a fine sintered glass filter, previously weighed. Dry the filter at 110 for 3 h, cool to room temperature, and weigh the filter. Calculate the amount of water-insoluble material.
for 15 min. Pass through a fine sintered glass filter, previously weighed. Dry the filter at 110 for 3 h, cool to room temperature, and weigh the filter. Calculate the amount of water-insoluble material.
Acceptance criteria:
NMT 10% of the amount of Extract taken
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight containers, and store at 25, excursion permitted between 15 and 30. Protect from light.
, excursion permitted between 15 and 30. Protect from light.
• Labeling:
The label states the Latin binomial and, following the official name of the article, the part of the plant from which the article was prepared, in addition to the information required for Botanical Extracts 565, Labeling.
565, Labeling.
• USP Reference Standards 11
11
USP Maritime Pine Extract RS
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Maged H. Sharaf, Ph.D.
Principal Scientific Liaison 1-301-816-8318 |
(DS2010) Monographs - Dietary Supplements |
| 2021 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 2022 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1381
Pharmacopeial Forum: Volume No. 32(4) Page 1142