Powdered Bilberry Extract
» Powdered Bilberry Extract is prepared from the ripe fruits of Vaccinium myrtillus L. (Fam. Ericaceae) using suitable solvents such as alcohol, methanol, or water or mixtures of these solvents. The ratio of the starting plant material to Powdered Extract is between 153:1 and 76:1. It contains not less than 36.0 percent of anthocyanosides, calculated as cyanidin-3-O-glucoside chloride, and not more than 1.0 percent of anthocyanidins, calculated as cyanidin chloride; both are calculated on the anhydrous basis.
Packaging and storage— Preserve in well-closed containers, protected from light and moisture, and store at controlled room temperature.
Labeling— The label states the Latin binomial and, following the official name, the part of the plant contained in the article. It meets other labeling requirements under Botanical Extracts 565.
Identification—
Standard solution— Dissolve about 100 mg of USP Powdered Bilberry Extract RS in 25 mL of methanol, centrifuge, and use the clear supernatant.
Test solution— Proceed as directed for the Standard solution, except to use Powdered Extract.
Adsorbent— Use suitable thin-layer chromatographic plates coated with a layer of cellulose.
Application volume: 10 µL.
Developing solvent system A— Use a mixture of water, glacial acetic acid, and hydrochloric acid (82:15:3).
Developing solvent system B— Use a mixture of glacial acetic acid and water (6:4).
Procedure— Use a saturated chamber. Develop the chromatograms using Developing solvent system A until the solvent front has moved about three-fourths of the plate, and dry the plate with the aid of a current of warm air. Develop the chromatograms in the same direction using Developing solvent system B until the solvent front has moved about three-fourths of the plate, and dry the plate with the aid of a current of warm air. Examine the plate under visible light: the chromatogram of the Test solution exhibits three main red bands with RF values of approximately 0.55, 0.65, and 0.70 that are similar in position and color to the corresponding main bands in the chromatogram of the Standard solution.
B: The retention times of the anthocyanoside peaks in the chromatogram of the Test solution correspond to those in the chromatogram of Standard solution 3, as obtained in the test for Content of anthocyanosides and anthocyanidins; the peaks due to delphinidin-3-O-galactoside chloride and delphinidin-3-O-glucoside chloride are the most intense peaks in the chromatogram and are of similar intensity, and each is more intense than the peak due to cyanidin-3-O-glucoside chloride; the peaks due to cyanidin-3-O-galactoside chloride, delphinidin-3-O-arabinoside chloride, and cyanidin-3-O-glucoside chloride are of similar intensity; and each of the remaining anthocyanoside peaks in the chromtogram is of lower intensity than the peak due to cyanidin-3-O-glucoside chloride.
Microbial enumeration 2021 The total aerobic microbial count does not exceed 104 cfu per g. The total combined yeasts and molds count does not exceed 103 cfu per g.
Absence of specified microorganisms 2022 It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
Water, Method Ia 921: not more than 4.5%, determined on 0.5 g.
Residue on ignition 281: not more than 3.0%, determined on 1.0 g.
Pesticide residues 561: meets the requirements.
Heavy metals, Method II 231: not more than 20 µg per g.
0.1 N Hydrochloric acid insoluble fraction— Finely grind about 5 g of Powdered Extract. Transfer about 1 g, accurately weighed, to a 500-mL flask, add 200 mL of 0.1 N hydrochloric acid, and shake vigorously for two hours. Filter the solution through a previously tared sintered-glass filter, wash the flask with 30 mL of 0.1 N hydrochloric acid, and transfer the washings to the filter. Wash the filter with 30 mL of 0.1 N hydrochloric acid in 5-mL portions. Dry the filter for three hours at 105, cool in a desiccator, and weigh. Calculate the percentage of the 0.1 N hydrochloric acid insoluble fraction: not more than 5% is found.
Content of anthocyanosides and anthocyanidins—
Solvent: a mixture of methanol and hydrochloric acid (98:2).
Diluent: a mixture of water and 85% phosphoric acid (9:1).
Solution A— Prepare a filtered and degassed mixture of water and formic acid (9:1).
Solution B— Prepare a filtered and degassed mixture of water, acetonitrile, methanol, and formic acid (40:22.5:22.5:10).
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 1— Dissolve, using sonication, an accurately weighed quantity of USP Cyanidin-3-O-glucoside Chloride RS in Solvent to obtain a solution having a known concentration of about 0.4 mg per mL. Transfer 2.0 mL of this solution to a 10-mL volumetric flask, dilute with Diluent to volume, and mix. [note—This solution is stable for 48 hours at 4.]
Standard solution 2— Dissolve, using sonication, an accurately weighed quantity of USP Cyanidin Chloride RS in Solvent to obtain a solution having a known concentration of about 0.5 mg per mL. Transfer 2.0 mL of this solution to a 100-mL volumetric flask, dilute with Diluent to volume, and mix. [note—This solution is stable for 36 hours at 4.]
Standard solution 3— Transfer about 125 mg of USP Powdered Bilberry Extract RS, accurately weighed, to a 100-mL volumetric flask, add 25 mL of Solvent, sonicate to dissolve, dilute with Diluent to volume, and mix. [note—This solution is stable for 48 hours at 4.]
Test solution— Proceed as directed for Standard solution 3, except to use Powdered Extract.
Chromatographic system (see Chromatography 621)— [note—Use deactivated silanized HPLC vials.] The liquid chromatograph is equipped with a refrigerated autosampler maintained at 4, a 535-nm detector, and a 4.6-mm × 25-cm column that contains 5-µm L1 packing. The flow rate is about 1.0 mL per minute. The column temperature is maintained at 30 ± 1. The chromatograph is programmed as follows:
Time
(minutes)
Solution A
(%)
Solution B
(%)
Elution
0–35 93®75 7®25 linear gradient
35–45 75®35 25®65 linear gradient
45–46 35®0 65®100 linear gradient
46–50 0 100 isocratic
50–51 0®93 100®7 linear gradient
51–60 93 7 isocratic
Chromatograph Standard solution 1, and record the peak responses as directed for Procedure: the tailing factor for the cyanidin-3-O-glucoside chloride peak is not less than 0.8 and not more than 2.0; and the relative standard deviation determined from the cyanidin-3-O-glucoside chloride peak for replicate injections is not more than 2.0%. Chromatograph Standard solution 3, and record the peak responses as directed for Procedure: the chromatogram obtained is similar to the Reference chromatogram provided with the lot of USP Powdered Bilberry Extract RS being used; the resolution, R, for the delphinidin-3-O-arabinoside, malvidin-3-O-galactoside, and petunidin-3-O-arabinose peaks is not less than 0.8; and the resolution, R, for other components is not less than 1.0.
Procedure— Separately inject equal volumes (about 10 µL) of Standard solution 1, Standard solution 2, Standard solution 3, and the Test solution into the chromatograph; record the chromatograms; and measure the areas of the analyte peaks. Using the chromatogram of Standard solution 3 and the Reference chromatogram provided with the lot of USP Powdered Bilberry Extract RS being used, identify the retention times of the peaks corresponding to the different anthocyanosides and anthocyanidins. The approximate relative retention times of the anthocyanosides and anthocyanidins are provided in the following table:
Analyte Relative
RetentionTime
Delphinidin-3-O-galactoside chloride 0.61
Delphinidin-3-O-glucoside chloride 0.73
Cyanidin-3-O-galactoside chloride 0.84
Delphinidin-3-O-arabinoside chloride 0.86
Cyanidin-3-O-glucoside chloride 1.00
Petunidin-3-O-galactoside chloride 1.08
Cyanidin-3-O-arabinoside chloride 1.11
Petunidin-3-O-glucoside chloride 1.24
Delphinidin chloride 1.28
Peonidin-3-O-galactoside chloride 1.36
Petunidin-3-O-arabinoside chloride 1.39
Peonidin-3-O-glucoside chloride 1.55
Malvidin-3-O-galactoside chloride 1.58
Peonidin-3-O-arabinoside chloride 1.67
Malvidin-3-O-glucoside chloride 1.76
Cyanidin chloride 1.82
Malvidin-3-O-arabinoside chloride 1.91
Petunidin chloride 2.08
Peonidin chloride 2.27
Malvidin chloride 2.30
Separately calculate the percentages of delphinidin-3-O-galactoside chloride, delphinidin-3-O-glucoside chloride, cyanidin-3-O-galactoside chloride, delphinidin-3-O-arabinoside chloride, cyanidin-3-O-glucoside chloride, petunidin-3-O-galactoside chloride, cyanidin-3-O-arabinoside chloride, petunidin-3-O-glucoside chloride, peonidin-3-O-galactoside chloride, petunidin-3-O-arabinoside chloride, peonidin-3-O-glucoside chloride, malvidin-3-O-galactoside chloride, peonidin-3-O-arabinoside chloride, malvidin-3-O-glucoside chloride, and malvidin-3-O-arabinoside chloride in the portion of Powdered Extract taken using the formula:
10,000(C/W)(rU / rS)
in which C is the concentration, in mg per mL, of USP Cyanidin-3-O-glucoside Chloride RS in Standard solution 1; W is the weight, in mg, of Powdered Extract taken to prepare the Test solution; rU is the peak response obtained for each of the anthocyanosides in the Test solution; and rS is the peak response obtained for cyanidin-3-O-glucoside chloride in Standard solution 1. Add the percentages calculated for all the anthocyanosides: not less than 36.0% is found. Separately calculate the percentages of delphinidin chloride, cyanidin chloride, petunidin chloride, peonidin chloride, and malvidin chloride in the portion of Powdered Extract taken using the formula:
10,000(C/W)(rU / rS)
in which C is the concentration, in mg per mL, of USP Cyanidin Chloride RS in Standard solution 2; W is the weight, in mg, of Powdered Extract taken to prepare the Test solution; rU is the peak response obtained for each of the anthocyanidins in the Test solution; and rS is the peak response obtained for cyanidin chloride in Standard solution 2. Add the percentages calculated for all the anthocyanidins: not more than 1.0% is found.
Other requirements— It meets the requirements of the test for Residual Solvents under Botanical Extracts 565.
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
2021 Radhakrishna S Tirumalai, Ph.D.
Senior Scientist
1-301-816-8339
(MSA05) Microbiology and Sterility Assurance
2022 Radhakrishna S Tirumalai, Ph.D.
Senior Scientist
1-301-816-8339
(MSA05) Microbiology and Sterility Assurance
USP32–NF27 Page 964
Pharmacopeial Forum: Volume No. 33(4) Page 685
Chromatographic Column—
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.