Powdered Hawthorn Leaf with Flower
DEFINITION
Powdered Hawthorn Leaf with Flower is Hawthorn Leaf with Flower reduced to a fine or very fine powder.
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identification Test 201
Standard solution:  0.1 mg/mL each of chlorogenic acid, rutin, USP Hyperoside RS, and USP Vitexin RS in methanol. [Note—Reserve a portion of this solution for use in Identification test B. ]
Sample solution:  Transfer about 1 g of Powdered Hawthorn Leaf with Flower to a flask, and add 10 mL of methanol. Heat the flask on a water bath maintained at 65 for 5 min, cool, filter, and use the filtrate.
Adsorbent:  0.50-mm layer of chromatographic silica gel mixture
Developing solvent system:  Ethyl acetate, glacial acetic acid, formic acid, and water (10:1.1:1.1:2.6)
Spray reagent A:  2-Aminoethyl diphenylborinate in methanol (1 in 100)
Spray reagent B:  Polyethylene glycol 4000 in methanol (5 in 100)
Analysis 
Samples:  Standard solution and Sample solution
Proceed as directed in the chapter, except to dry the plate at 105, and spray the plate while still hot with 10 mL of Spray reagent A and then with 10 mL of Spray reagent B. Allow the plate to air-dry for 30 min, and examine the plate under long-wavelength UV light.
Acceptance criteria:  The chromatogram of the Standard solution exhibits an intense orange zone (at RF value of 0.3) due to rutin; a light blue fluorescent zone (at RF value of 0.4) due to chlorogenic acid; a yellowish-orange zone (at RF value of 0.55) due to hyperoside; and a yellowish-green zone (at RF value of 0.65) due to vitexin. The chromatogram of the Sample solution, in addition to the zones due to rutin, chlorogenic acid, hyperoside, and vitexin, exhibits a yellowish-green zone (at RF value of 0.35) due to vitexin-2-rhamnoside; a light blue fluorescent zone (at RF value of 0.6) due to spiraeoside; and a light blue fluorescent zone near the solvent front (at RF value of 0.9) due to caffeic acid. The chromatogram of the Sample solution also exhibits other zones with weaker fluorescence.
•  B. Procedure
Solution A:  Tetrahydrofuran, acetonitrile, and methanol (92.4:3.4:4.2)
Solution B:  0.5% phosphoric acid in water
Mobile phase:  See the gradient table below.
Time
(min)
Solution A
(%)
Solution B
(%)
0 12 88
12 12 88
25 18 82
30 18 82
Standard solution:  Use the Standard solution reserved from Identification test A.
Sample solution:  Transfer about 3 g of Powdered Hawthorn Leaf with Flower to a 100-mL round-bottom flask, add 60 mL of a mixture of methanol and water (4:1), and heat on a hot water bath under reflux for 1 h. Cool, filter, and collect the filtrate in a separate flask. Transfer the residue from the filter back to the flask, add 40 mL of a mixture of methanol and water (4:1), and heat on a hot water bath under reflux for 10 min. Cool, filter, and combine the filtrate with the filtrate obtained from the first extraction. Evaporate the solvent from the combined filtrates under vacuum to a volume of 20 mL. Dilute the resulting solution with a mixture of methanol and water (4:1) to 25.0 mL. Filter 5.0 mL of this solution through a freshly conditioned solid-phase extraction column containing 360 mg of packing L1, collect the filtrate in a 10-mL volumetric flask, and dilute with a mixture of methanol and water (4:1) to volume.
Chromatographic system 
Mode:  LC
Detector:  UV 336 nm
Column:  4.0-mm × 10-cm; 5-µm packing L1
Temperature:  25
Flow rate:  1 mL/min
Injection size:  5 µL
System suitability 
Sample:  Standard solution
[Note—The relative retention times for chlorogenic acid, vitexin, rutin, and hyperoside are 0.26, 1.0, 1.16, and 1.4, respectively. ]
Suitability requirements 
Relative standard deviation:  NMT 2.0%
Analysis 
Samples:  Standard solution and Sample solution
[Note—The relative retention times for acetyl vitexin-2¢¢-O-rhamnoside, vitexin, isovitexin, and vitexin-2¢¢-O-rhamnoside are 1.53, 1.0, 0.73, and 0.67, respectively. ]
Measure the retention times for the major peaks.
Acceptance criteria:  The retention times of the peaks for chlorogenic acid, vitexin, rutin, and hyperoside of the Sample solution correspond to those of the Standard solution.
COMPOSITION
•  Content of C-Glycosylated Flavones
Solution A:  0.5% solution of phosphoric acid in water
Solution B:  Tetrahydrofuran, isopropyl alcohol, and acetonitrile (10:8:3)
Mobile phase:  Solution A and Solution B (22:3)
Standard solution:  0.3 mg/mL of USP Vitexin RS in Solution B, with heating if necessary
Sample solution:  Transfer about 4 g of Powdered Hawthorn Leaf with Flower, accurately weighed, to a continuous-extraction apparatus fitted with a flask containing 80 mL of methanol, and extract the test specimen for 5 h. Cool, remove the flask, and evaporate the solvent from the extract under vacuum to 40 mL. Transfer this solution to a 50-mL volumetric flask, and dilute with methanol to volume. Transfer 10.0 mL of the solution to a suitable flask fitted with a reflux condenser, add 4 mL of 25% hydrochloric acid, and heat the flask under reflux on a water bath at 65 for 90 min. Cool, transfer the contents of the flask to a 50-mL volumetric flask, and dilute with methanol to volume.
Chromatographic system 
Mode:  LC
Detector:  UV 336 nm
Column:  4-mm × 10-cm; packing L1
Flow rate:  1 mL/min
Injection size:  5 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Column efficiency:  NLT 3000 theoretical plates
Tailing factor:  0.8–2
Relative standard deviation:  NMT 2.0%
Analysis 
Samples:  Standard solution and Sample solution
Measure the areas of the major peaks. Calculate the percentage of C-glycosylated flavones, expressed as vitexin (C21H20O10), in the portion of Powdered Hawthorn Leaf with Flower taken:
Result = (SrU/rS) × (CS/CU) × 100
SrU== sum of the peak areas of vitexin and isovitexin, with a relative retention time of about 1.0 and 0.85, respectively, from the Sample solution
rS== vitexin peak area from the Standard solution
CS== concentration of USP Vitexin RS in the Standard solution (mg/mL)
CU== concentration of Powdered Hawthorn Leaf with Flower in the Sample solution (mg/mL)
Acceptance criteria:  NLT 0.6% of C-glycosylated flavones, expressed as vitexin (C21H20O10)
•  Content of O-Glycosylated Flavones
Mobile phase:  Methanol, phosphoric acid, and water (100:1:100)
Standard solution:  0.05 mg/mL of USP Quercetin RS in methanol
Sample solution:  Proceed as directed for the Sample solution under Content of C-Glycosylated Flavones, except to use 1 mL of 25% hydrochloric acid for 60 min instead of 4 mL of 25% hydrochloric acid for 90 min.
Chromatographic system 
Mode:  LC
Detector:  UV 370 nm
Column:  4.6-mm × 25-cm; packing L1
Flow rate:  1.5 mL/min
Injection size:  10 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Column efficiency:  NLT 3000 theoretical plates
Tailing factor:  0.8–2
Relative standard deviation:  NMT 2.0%
Analysis 
Samples:  Standard solution and Sample solution
Measure the areas of the major peaks. Calculate the percentage of O-glycosylated flavones, expressed as hyperoside (C21H20O12), in the portion of Powdered Hawthorn Leaf with Flower taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
rU== quercetin peak area from the Sample solution
rS== quercetin peak area from the Standard solution
CS== concentration of USP Quercetin RS in the Standard solution (mg/mL)
CU== concentration of Powdered Hawthorn Leaf with Flower in the Sample solution (mg/mL)
Mr1== molecular weight of hyperoside, 464.38
Mr2== molecular weight of quercetin, 302.24
Acceptance criteria:  NLT 0.45% of O-glycosylated flavones, expressed as hyperoside (C21H20O12)
IMPURITIES
Inorganic Impurities 
•  Heavy Metals, Method III 231: NMT 20 ppm
Organic Impurities 
•  Procedure 1: Articles of Botanical Origin, Foreign Organic Matter 561: NMT 8.0% of lignified matter
•  Procedure 2: Articles of Botanical Origin, Pesticide Residues 561: Meets the requirements
SPECIFIC TESTS
•  Botanic Characteristics
Microscopic:  When examined under a microscope, the yellowish-green powder shows the following characteristics: unicellular covering trichomes, usually with thick walls and wide lumens, almost straight to somewhat curved, pitted at the base; fragments of leaf epidermis with cells that have sinuous to polygonal walls and large anomocytic stomata surrounded by four to seven subsidiary cells; clusters of parenchymatous cells containing calcium oxalate crystals, usually from 10–20 µm in length; fragments of petals showing rounded polygonal epidermal cells, strongly papillose, with thick walls, the cuticle of which clearly shows wavy striations; fragments of anthers whose endothecium has an arched and regularly thickened margin; fragments of stems containing collenchymatous cells, vessels, and fibers of lignified sclerenchyma, with narrow lumens; and numerous rounded to elliptical triangular pollen grains up to 45 µm in diameter, with free exines and three germinal pores.
•  Microbial Enumeration Tests—Nutritional and Dietary Supplements 2021: The total bacterial count does not exceed 104 cfu/g, the total combined molds and yeasts count does not exceed 100 cfu/g, and it meets the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus.
•  Loss on Drying 731: Dry about 1.0 g of Powdered Hawthorn Leaf with Flower at 105 for 2 h: it loses NMT 10.0% of its weight.
ADDITIONAL REQUIREMENTS
•  Packaging and storage: Store in a well-closed container, protected from light.
•  Labeling: The label states the Latin binomial and, following the official name, the parts of the plant source from which the article was derived.
•  USP Reference Standards 11
USP Hyperoside RS
USP Quercetin RS
USP Vitexin 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
Reference Standards RS Technical Services
1-301-816-8129
rstech@usp.org
USP35–NF30 Page 1356
Pharmacopeial Forum: Volume No. 34(5) Page 1209