St. John's Wort
DEFINITION
St. John's Wort consists of the dried flowering tops or aerial parts of Hypericum perforatum L. (Fam. Hypericaceae), gathered shortly before or during flowering. It contains NLT 0.04% of the combined total of hypericin (C30H16O8) and pseudohypericin (C30H16O9) and NLT 0.6% of hyperforin (C35H52O4), on the dried basis.
IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test
Standard solution:
0.5 mg/mL of USP Hyperoside RS in methanol
Sample solution:
Finely powder 50 g of St. John's Wort. Shake 10 g in 100 mL of methanol for about 15 min, and filter.
Chromatographic system
Adsorbent:
0.50-mm layer of chromatographic silica gel mixture
Application volume:
20 µL
Developing solvent system:
Upper layer of a mixture of ethyl acetate, glacial acetic acid, formic acid, and water (10: 1.1: 1.1: 2.6)
Spray reagent A:
10-mg/mL solution of 2-aminoethyl diphenylborinate in methanol
Spray reagent B:
50-mg/mL solution of polyethylene glycol 400 in alcohol
Analysis
Samples:
Standard solution and Sample solution
Apply the Samples as bands and allow to dry. Develop and allow the plate to air-dry. Spray with Spray reagent A and allow the plate to air-dry. Immediately after, spray the plate with Spray reagent B, and allow the plate to air-dry. Examine the plate under UV light at 365 nm.
Acceptance criteria:
The chromatogram of the Sample solution exhibits several zones having a yellowish-orange fluorescence, one of which, appearing at an RF value of about 0.5, corresponds in RF value and intensity to a similar zone in the chromatogram of the Standard solution. The chromatogram of the Sample solution exhibits also two zones of red fluorescence, one at an RF value of about 0.85 (presence of hypericin) and the other at an RF value of about 0.80 (presence of pseudohypericin), and two zones of higher blue fluorescence (presence of chlorogenic and neochlorogenic acids) located below the yellow to yellowish-orange hyperoside zone.
COMPOSITION
• Content of Hypericin and Pseudohypericin
[Note—Conduct all sample preparations with minimal exposure to subdued light, and use low-actinic glassware to protect solutions from light. ]
Solvent:
Methanol and acetone (1:1)
Solution A:
Phosphoric acid and water (3:997)
Solution B:
Acetonitrile
Solution C:
Methanol
Mobile phase:
See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
Solution C (%) |
|---|---|---|---|
| 0 | 100 | 0 | 0 |
| 10 | 85 | 15 | 0 |
| 30 | 70 | 20 | 10 |
| 40 | 10 | 75 | 15 |
| 55 | 5 | 80 | 15 |
| 56 | 100 | 0 | 0 |
| 66 | 100 | 0 | 0 |
Standard solution A:
2.5 µg/mL of USP Oxybenzone RS in Solvent
Standard solution B:
1 mg/mL of USP Powdered St. John's Wort Extract RS in Solvent
Sample solution:
Weigh and pulverize 10 g of St. John's Wort. Transfer 1 g to a round-bottom flask equipped with a condenser and protected from light, add 50 mL of Solvent and a magnetic stirring bar, and heat at 60
for 2 h while stirring. Cool to room temperature, and pass through filter paper into a 50-mL volumetric flask. Wash the flask and the residue on the filter with Solvent, and dilute with the washings to volume. Pass the solution through a polytef membrane filter having a 0.45-µm or finer pore size, and use the filtrate.
for 2 h while stirring. Cool to room temperature, and pass through filter paper into a 50-mL volumetric flask. Wash the flask and the residue on the filter with Solvent, and dilute with the washings to volume. Pass the solution through a polytef membrane filter having a 0.45-µm or finer pore size, and use the filtrate.
Chromatographic system
Mode:
LC
Detector:
UV 270 nm and Vis 588 nm
Columns
Guard:
Packing L1
Analytical:
4.6-mm × 25-cm; packing L1
Column temperature:
30
Flow rate:
1 mL/min
Injection size:
20 µL. [Note—First equilibrate the system with 100% Solution A. ]
System suitability
Samples:
Standard solution A (record the peak responses at 270 nm), and Standard solution B (record the peak responses at 270 nm and 588 nm)
Suitability requirements
Chromatogram similarity:
The chromatograms from Standard solution B are similar to the respective reference chromatograms provided with the lot of USP Powdered St. John's Wort Extract RS being used.
Column efficiency:
NLT 100,000 theoretical plates for oxybenzone, Standard solution A
Tailing factor:
NMT 1.5 for oxybenzone, Standard solution A
Relative standard deviation:
NMT 2.0%, Standard solution A
Analysis
Samples:
Standard solution A and Sample solution
Measure the areas of the relevant peaks at 270 nm in the chromatogram of the Sample solution.
Separately calculate the percentage of hypericin (C30H16O8) and pseudohypericin (C30H16O9) in the portion of St. John's Wort taken:
Result = (rU/rS) × CS × (V/W) × F × 100
| rU | = | = peak area of the relevant analyte from the Sample solution |
| rS | = | = peak area of oxybenzone from Standard solution A |
| CS | = | = concentration of USP Oxybenzone RS in Standard solution A (mg/mL) |
| V | = | = volume of the Sample solution (mL) |
| W | = | = weight of St. John's Wort taken to prepare the Sample solution (mg) |
| F | = | = relative response factor for the relevant analyte relative to that of oxybenzone, 1.30 for hypericin and 1.24 for pseudohypericin |
Calculate the combined total of hypericin and pseudohypericin by adding the corresponding percentages as calculated above.
Acceptance criteria:
NLT 0.04% on the dried basis
• Content of Hyperforin
Analysis:
Using the chromatograms obtained in the test for Content of Hypericin and Pseudohypericin, calculate the percentage of hyperforin (C35H52O4) in the portion of St. John's Wort taken:
Result = (rU/rS) × CS × (V/W) × F × 100
| rU | = | = peak area of hyperforin from the Sample solution |
| rS | = | = peak area of oxybenzone from Standard solution A |
| CS | = | = concentration of USP Oxybenzone RS in Standard solution A (mg/mL) |
| V | = | = volume of the Sample solution (mL) |
| W | = | = weight of St. John's Wort taken to prepare the Sample solution (mg) |
| F | = | = response factor for hyperforin relative to that of oxybenzone, 0.46 |
Acceptance criteria:
NLT 0.6% on the dried basis
CONTAMINANTS
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 
561
:
Meets the requirements
561:
Meets the requirements
• Microbial Enumeration Tests 2021:
The total bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 102 cfu/g.
2021:
The total bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 102 cfu/g.
• Absence of Specified Microorganisms 2022:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli and for absence of Staphylococcus aureus.
2022:
It meets the requirements of the tests for absence of Salmonella species and Escherichia coli and for absence of Staphylococcus aureus.
SPECIFIC TESTS
• Botanic Characteristics
Macroscopic:
The two-edged stem is greenish yellow, rounded, and has two ribs running longitudinally on opposite sides. The plant is adversifoliate, its leaves are sessile, ovoid or elongated, up to 3.5 cm in length, smooth-edged, and hairless with translucent perforations. The very numerous yellow, short-stemmed, pentamerous flowers form false umbels shaped like grape clusters. The five lanceolate and black-dotted sepals are about one-half the length of the dark yellow petals, which are shaped like slanted ovals and whose edges are set with dark red glands. The numerous stamens are joined in three to six bundles (usually three). The ovary is surmounted by three styles. Some ovaries are already developed into greenish, elongated, oval triovular capsules with various degrees of maturity. When chopped, the crude plant material is distinguished by numerous yellow to yellowish-brown flower buds and individual petals with dark red glands at the edges. The light green to brown-green leaf fragments, characterized by plicate marcescence, appear stippled when held up to the light. The greenish yellow or reddish-brown hollow stem fragments are distinguished by two longitudinal edges.
Microscopic:
The stems have elongated epidermal cells with straight beaded, anticlinal walls; cuticle smooth; frequent paracytic stomata with two small adjacent epidermal cells; cortex of 5–6 rows of collenchyma; stele with secondary growth consisting of a compacted ring of phloem, with a wide area of lignified xylem and small areas of intraxylary phloem; parenchymatous pith, lignified and pitted in older stems; oil glands may occur in the cortex and phloem.
The upper surface of the leaf has polygonal cells with sinuous, slightly beaded anticlinal walls; cells of lower surface smaller, with anticlinal walls more wavy with frequent paracytic, sometimes anomocytic, stomata; smooth cuticle, thicker on upper surface, straight-walled, elongated epidermal cells of veins occasionally beaded. Dorsiventral, single palisade lamina; large oil glands equal to depth of spongy mesophyll. Midrib containing single, collateral bundle with small area of lignified xylem. Trichomes and calcium oxalate are absent.
The sepal of the flower has characteristics resembling those of the leaf. Petal, narrow, elongated, thin-walled, epidermal cells with straight anticlinal walls on the outer surface and wavy on the inner surface. Stamen, lignified fibrous layer of anther wall; elongated, thin-walled cells of filament with striated cuticle; subprolate pollen grains, about 20 µm in diameter with three pores and a smooth exine. Ovary, small polygonal cells with underlying oil glands; seed testa, brown, thick-walled hexagonal cells.
• Articles of Botanical Origin, Total Ash 561:
NMT 5.0%
561:
NMT 5.0%
• Articles of Botanical Origin, Water Content 561:
NMT 10.0%
561:
NMT 10.0%
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Store in tight containers, protected from light and moisture.
• Labeling:
The label states the Latin binomial and, following the official name, the parts of the plant contained in the article.
• USP Reference Standards 11
11
USP Hyperoside RS
USP Oxybenzone RS 
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USP Powdered St. John's Wort Extract RS
USP Rutin RS
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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 1426