Cullen corylifolium Fruit

Cullen corylifolium Fruit

Proposed For Development Version 0.1

Cullen corylifolium Fruit

 


 

DEFINITION

 

The article consists of the dried fruit of Cullen corylifolium (L.) Medik., formerly known as Psoralea corylifolia (Family Fabaceae). The fruit may be misidentified as seeds. It contains NLT 0.2% of psoralen on the dried basis.

 

SYNONYMS

Cullen corylifolia (L.) Medik.

Cullen corylifolius (L.) Medik.

Lotodes corylifolia (L.) Kuntze

Psoralea corylifolia L.

Psoralea patersoniae Schonl.

Trifolium unifolium Forssk.

 

POTENTIAL CONFOUNDING MATERIALS

Senna tora (L.) Roxb.

 

SELECTED COMMON NAMES

Chinese: 补骨脂

English: Blackdot, psoralea, scurfy-pea

Hindi: Babchi, bavachi, bakuchi

Swedish: Skovärt

 

CONSTITUENTS OF INTEREST

Furocoumarin: Psoralen

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Single seeded fruits, with thin papery pericarp, almost inseparable from the seed, closely attached to the seed coat. Though the calyx is not persistent, rarely, unequal pale colored translucent calyx lobes are found attached in a few fruits. Dark brown to light black, dull, with rough surface. They are variously or irregularly shaped, shape range from round or ovoid–oblong–mucronate, even bean shaped, laterally flattened or compressed, and around 4.5 × 3 mm in size. The surface is deeply wrinkled or shrunken, having irregularly running ridges with furrows, and finely reticulated or pitted. The seeds are reniform, brown or dark brown in color, 2–3 × 1–1.5 mm in size, with smooth surface and with straw to brown colored hard testa. The seed consists of two elongated somewhat kidney-shaped oily cotyledons.

Microscopic: The cross section of the fruit is oval to circular, with prominent, high raised ridges and depressions in the outline. The outermost cover comprises outer pericarp, followed by testa, embryo and cotyledons in the center. The outermost layer resembles cork cells. Inner to this is a highly collapsed or compressed thin-walled parenchyma cells. Some pericarp cells are filled with dark contents. Adjacent to the pericarp is the testa made of longitudinally elongated macrosclereids and measuring about 44–54 µm long, followed by a layer of osteosclereids measuring about 11–20 µm in length. Below this region are a few layers of thick-walled and or collapsed parenchyma cells followed by a layer of tegmen made up of thick-walled, compactly arranged cells some containing brown pigment. Cotyledons show outer epidermis and inner ground tissue. The epidermal cells are smaller and compactly arranged in a single row. The ground tissue is made of compactly packed palisade-like cells and spongy parenchyma cells. The palisade cells are elongated, arranged in 2–3 rows, just below the epidermis, towards the commissural sides of the cotyledons, measureing about 46–60 µm in length. The vascular traces are clearly visible between these two layers. Oil globules are commonly observed in the cotyledon cells.

 

B. HPTLC for Identification of Articles of Botanical Origin <203>

Standard solution A: 1.0 mg/mL of USP Psoralen RS in methanol

Standard solution B: 50 mg/mL of USP Cullen corylifolium Fruit Dry Extract RS in methanol. Sonicate for 10 min, centrifuge or filter, and use the supernatant or the filtrate.

Sample solution: Sonicate about 1 g of Cullen corylifolium Fruit, finely powdered, in 10 mL of methanol and reflux for 15 min, centrifuge or filter, and use the supernatant.

Chromatographic system

Adsorbent: Chromatographic silica gel F254 mixture with an average particle size of 5 µm.

Application volume: 5 µL of Standard solution A, 2 µL of Standard solution B, and 5 µL of the Sample solution, as 8-mm bands

Relative humidity: Condition the plate to a relative humidity of about 33% using a suitable device.

Developing solvent system: Toluene, ethyl acetate, and formic acid (80:18:2)

Developing distance: 6 cm

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

  Apply the Samples as bands and dry in air. Develop in a saturated chamber (20 min with filter paper), remove the plate from the chamber, and dry in air. Examine under UV light at 254 and 365 nm.

System suitability: Under UV 254 nm, the chromatogram of Standard solution B exhibits one intense black band and two black bands in upper half of the chromatogram, with the lower black band near the middle of the chromatogram similar in RF and color to the psoralen band in the chromatogram of Standard solution A. About eight additional black bands seen in the lower half of the chromatogram. One bluish band appears near RF 0.3. Under UV 365 nm, a blue fluorescent band appears in the upper half of the chromatogram similar in RF and color to the psoralen band in the chromatogram of Standard solution A. A black band, faint blue band, and two orange bands, with increasing RF above the psoralen band appear in Standard solution B. A bright blue fluorescent band and a less bright fluorescent band appear below the psoralen band. Below the less fluorescent band, with decreasing RF, a dark intense black band and an intense bright blue fluorescent band appear in Standard solution B.

Acceptance criteria: Under UV 254 nm, the chromatogram of the Sample solution exhibits one black band near the middle of the chromatogram corresponding in RF and color to the psoralen band in the chromatogram of Standard solution A. Two additional black bands appear above the psoralen band, with an intense black band in the upper RF. About eight black bands in the lower half of the chromatogram with one bluish black band near RF 0.3. Under UV 365 nm, a blue band similar in RF and color to the psoralen band in the chromatogram of Standard solution A. Above the psoralen band, with increasing RF, a faint white or bluish band, two orange bands appear. Below the psoralen band, with decreasing RF, two blue bands, a black band, a bright blue band, and several blue bands appear.

 

• C. HPLC

Analysis: Proceed as directed in the test for Content of Psoralen.

Acceptance criteria: The chromatogram of the Sample solution exhibits peaks at retention times corresponding to the peak due to psoralen in Standard solution B.

 

ASSAY

• Content of Psoralen

Solution A: 0.1% trifluoroacetic acid in water

Solution B: 0.1% trifluoroacetic acid in acetonitrile

Mobile phase: See Table 2.

 

Table 2

Time
(min)

Solution A
(%)

Solution B
(%)

0

90

10

5

85

15

8

70

30

15

40

60

22

40

60

25

10

90

30

10

90

32

90

10

35

90

10

 

Standard solution A: 1 mg/mL of USP Psoralen RS in methanol

Standard solution B: 5 mg/mL of USP Cullen corylifolium Fruit Dry Extract RS in methanol. Sonicate, and pass through a membrane filter of 0.45-µm or finer pore size.

Sample solution: Transfer 1 g of Cullen corylifolium Fruit, finely powdered and accurately weighed, to a 250-mL flask and add 70 mL of methanol. Reflux at 80° for 30 min and transfer the extract into a 100-mL volumetric flask. Repeat the extraction process with another 20 mL of methanol and add the extract to the volumetric flask. Dilute with methanol to volume. Pass through a membrane filter of 0.45-µm pore size.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Detector: UV 290 nm

Column: 4.6-mm × 10-cm; 2.7-µm packing L1

Column temperature: 40°

Flow rate: 1.0 mL/min

Injection volume: 10 µL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Chromatogram similarity: The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Cullen corylifolium Fruit Dry Extract RS being used.

Resolution: NLT 2.0 between the psoralen peak and the peak after, Standard solution B

Tailing factor: NMT 2.0 for the psoralen peak, Standard solution A

Relative standard deviation: NMT 2.0% for the psoralen peak in repeated injections, Standard solution A

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Using the chromatograms of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Cullen corylifolium Fruit Dry Extract RS being used, identify the retention times of the peaks corresponding to psoralen in the Sample solution.

Calculate the percentages of psoralen in the portion of Cullen corylifolium Fruit taken:

 

Result = (rU/rS) × CS × (V/W) × 100

 

rU   = peak area of psoralen from the Sample solution

rS   = peak area of psoralen from Standard solution A

CS  = concentration of USP Psoralen RS in Standard solution A (mg/mL)

V   = volume of the Sample solution (mL)

W   = weight of Cullen corylifolium Fruit taken to prepare the Sample solution (mg)

 

Acceptance criteria: NLT 0.2% on the dried basis

 

CONTAMINANTS

• Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 0.5 µg/g

Lead: NMT 5.0 µg/g

Mercury: NMT 0.2 µg/g

• Articles of Botanical Origin <561>, Pesticide Residue Analysis: Meets the requirements

• Microbial Enumeration Tests <61>: The total aerobic bacterial count does not exceed 105 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the bile-tolerant Gram-negative bacteria does not exceed 103 cfu/g.

• Tests for Specified Microorganisms <62>: Meets the requirements of the tests for the absence of Salmonella species and Escherichia coli

 

SPECIFIC TESTS

• Articles of Botanical Origin <561>, Methods of Analysis, Foreign Organic Matter: NMT 2.0%

• Articles of Botanical Origin <561>, Methods of Analysis, Alcohol-Soluble Extractives, Method 1: NLT 30.0%

• Articles of Botanical Origin <561>, Methods of Analysis, Water-Soluble Extractives, Method 2: NLT 10.0%

• Loss on Drying <731>

Sample: 2 g of Cullen corylifolium Fruit, finely powdered

Analysis: Dry the Sample at 105° for 5 h.

Acceptance criteria: NMT 10%

• Articles of Botanical Origin <561>, Methods of Analysis, Total Ash: NMT 101.0%

• Articles of Botanical Origin, Acid-Insoluble Ash <561>: NMT 2.0%

 

ADDITIONAL REQUIREMENTS

• Packaging and Storage: Preserve in well-closed containers, protected from light and moisture, and store at room temperature.

• Labeling: The label states the Latin binomial and the part(s) of the plant contained in the article.

• USP Reference Standards <11>

USP Cullen corylifolium Fruit Dry Extract RS

USP Psoralen RS

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