Trigonella foenum-graecum Seed

Trigonella foenum-graecum Seed

Proposed For Comment Version 0.2

Trigonella foenum-graecum Seed


 

DEFINITION

The article consists of the dried ripe seeds of Trigonella foenum-graecum L. (Family Fabaceae). It contains NLT 0.2% of 4-hydroxyisoleucine, calculated on the dried basis.

 

SYNONYMS

Trigonella foenum-graecum subsp. gladiata (M. Bieb.) P. Fourn.

 

POTENTIAL CONFOUNDING MATERIALS

None known

 

SELECTED COMMON NAMES

Arabic: حلبة

Chinese: 胡卢巴

Danish: Almindelig bukkehorn

English: Fenugreek, Greek hay, Greek clover, Greek hay seed

French: Fenugrec

German: Bockshornklee

Indian: Methi (Punjabi), menthe and mente (Kannada), uluva (Malayalam), mendium and ventaiyam (Tamil), mentulu (Telego)

Japanese: フェヌグリーク (fenugriiku), コロハ (koroha)

Korean: 호로파 (horopa)

Pinyin: Hu lu ba

Portugese: Feno-greco (Brazil)

Russian: пажитник греческий

Spanish: Alholva, heno griego, fenogreco

Swedish: Bockhornsklöver

 

CONSTITUENTS OF INTEREST

Amino acids: 4-Hydroxyisoleucine, 4-hydroxyisoleucine lactone, arginine, histidine, and lysine

Steroidal saponins: Trigoneoside IIa, Ib; graecunins H, I, J, K, L, M, N; trigofoenosides A, D, F, G; protogracillin; protodioscin; and diosgenin

Alkaloids: Trigonelline, gentianine, and carpaine

Flavonoids: Apigenin, luteolin, orientin, quercetin, vitexin, and isovitexin

Carbohydrates: Mainly mucilnage

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Seeds are oblong, flattened, or irregularly rhomboidal, with rounded edges; 3–5 mm long, 2–3 mm wide, about 2 mm thick; hard; yellowish-brown outside, yellow inside; nearly in the center of one of the long narrow sides is a small depression exhibiting a white point, the hilum, lying adjacent to the micropyle and raphae; the depression extends diagonally, in the form of a furrow on the widest surfaces and divides the seed into two unequal parts. The smaller part contains the radicle and the larger part contains the cotyledons.

Microscopic

Transverse cut: Shows yellowish embryo consisting of two large cotyledons encircled by dark, translucent endosperm filled with mucilage, and a crescent-shape radicle in a small pocket.

Transverse section: Testa—the outermost layer is covered with cuticle and composed of thick-wall, cylindrical, lignified cells with conical projections, through which a white line, linea lucida, extends across; middle layer of testa composed of cells with thick-walls and wide intercellular spaces near their tops; inner section of testa composed of a few layers of narrow, tangentially elongated, compact, thin-wall parenchyma cells. Endosperm—outer layer consists of rectangular to polygonal thick-wall cells, full of aleurone grains; multiple layers of cells of various shapes, large, full of mucilage. Cotyledons—a layer of epidermal cells with thick walls; 3–4 layers of cells containing oil globules; a few layers of spongy tissue.

B. Thin-Layer ChromatographyAmino Acids Profile

Standard solution A: 0.5 mg/mL of USP 4-Hydroxyisoleucine RS in methanol

Standard solution B: 50 mg/mL of USP Trigonella foenum-graecum Seed Powdered Extract RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Sample solution: About 1.0 g of Trigonella foenum-graecum Seed, finely powdered, in 5.0 mL of methanol, heat in a water bath at 65º for 5 min, and filter.

[NoteSave the Sample solution for use in Identification C and the Specific Test­s—Presence of Trigonelline.]

Chromatographic system

(See Chromatography <621>, Thin-Layer Chromatography.)

Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)

Application volume: 2 µL each of Standard solution A and Standard solution B, and 4 µL of Sample solution, as 8-mm bands

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

Temperature: 25°

Developing solvent system: n-Butanol, acetic acid, and water (7:2:1)

Developing distance: 6 cm

Derivatization reagent: Ninhydrin reagent – 0.3 g of ninhydrin, 95 mL of isopropanol, and 5 mL of glacial acetic acid.

Analysis

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

Apply the Samples as bands to a suitable HPTLC plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry. Treat with Derivatization reagent, heat for 2 min at 100º–105°, and examine under visible light and UV light at 366 nm.

System suitability: Under visible light, the chromatogram of Standard solution B exhibits, in the lower half, five brown bands in the following order of increasing RF: a minor band, the most intense band at an RF corresponding to the 4-hydroxyisoleucine band in the chromatogram of Standard solution A, and three less intense bands.

Under UV light at 366 nm, the chromatogram of Standard solution B exhibits, in the lower half, four bands in the following order of increasing RF: a minor dark brown band, the most intense band as a dark brown band at an RF corresponding to the 4-hydroxyisoleucine band in the chromatogram of Standard solution A, and two less intense purple bands. The chromatogram of Standard solution B exhibits, in the upper half, three yellow bands, the one with the lowest RF appears diffuse and, in cases, may appear resolved into two bands.

Acceptance criteria: Under visible light, the chromatogram of the Sample solution exhibits, in the lower half, the most intense band as a brown band at an RF corresponding to the 4-hydroxyisoleucine band in the chromatogram of Standard solution A, and four additional brown bands corresponding to similar bands in the chromatogram of Standard solution B: a minor band at an RF below that of the 4-hydroxyisoleucine, and three less intense bands above the 4-hydroxyisoleucine band.

Under UV light at 366 nm, the chromatogram of the Sample solution exhibits, in the lower half, the most intense band as a dark brown band at an RF corresponding to the 4-hydroxyisoleucine band in the chromatogram of Standard solution A, and three additional bands corresponding to similar bands in the chromatogram of Standard solution B: a minor dark brown band at an RF below that of the 4-hydroxyisoleucine, and two less intense purple bands above the 4-hydroxyisoleucine band. The chromatogram of the Sample solution exhibits, in the upper half, three yellow bands corresponding to similar bands in the chromatogram of Standard solution B.

C. Thin-Layer ChromatographySteroidal Saponins Profile

Standard solution A: 50 mg/mL of USP Trigonella foenum-graecum Seed Powdered Extract RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Sample solution: Use the Sample solution prepared in Identification B.

Chromatographic system

(See Chromatography <621>, Thin-Layer Chromatography.)

Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)

Application volume: 2 µL, as 8-mm bands

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

Temperature: 25°

Developing solvent system: Dichloromethane, methanol, and water (18:8:1)

Developing distance: 6 cm

Derivatization reagent: Anisaldehyde reagent – 85 mL of ice-cooled methanol mixed with 10 mL of glacial acetic acid, 5 mL of sulfuric acid, and 0.5 mL of p-anisaldehyde.

Analysis

Samples: Standard solution A and Sample solution

Apply the Samples as bands to a suitable HPTLC plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry. Treat with Derivatization reagent, heat for 3 min at 100°, and examine under visible light and UV light at 366 nm.

System suitability: Under visible light, the chromatogram of Standard solution A exhibits, in the lower half, four intense bands in the following order of increasing RF: a brown band, a brown band due to protogracillin, a brown band due to protodioscin, and a violet band. The chromatogram of Standard solution B exhibits, in the upper half, two violet bands and a third violet band close to the solvent front.

Under UV light at 366 nm, the chromatogram of Standard solution A exhibits, in the lower half, three blue fluorescent bands interspersed with three brown bands due to saponins, a pink band, and a broad blue fluorescent band. Acceptance criteria: Under visible light, the chromatogram of the Sample solution exhibits three intense brown bands and a violet band in the lower half, and three violet bands in the upper half. All of these bands correspond to similar bands in the chromatogram of Standard solution A.

Under UV light at 366 nm, the chromatogram of the Sample solution exhibits, in the lower half, three blue fluorescent bands interspersed with three brown bands, a pink band, and a broad blue fluorescent band. All of these bands correspond to similar bands in the chromatogram of Standard solution A.

 

ASSAY

• Content of 4-Hydroxyisoleucine

Solution A: 0.1% Phosphoric acid in water (v/v)

Solution B: Acetonitrile

Mobile phase: See Table 1.

 

Table 1

Time
(min)

Solution A
(%)

Solution B
(%)

0

80

20

20

40

60

 

Reagent: A mixture of acetonitrile, water, and triethylamine (40:12:8)

Diluent: Methanol and water (1:1) 

Standard solution: Transfer about 2.0 mg of USP 4-Hydroxyisoleucine RS, accurately weighed, to a 100-mL volumetric flask, dissolve in 10 mL of Reagent, add 500 µL of phenyl isothiocyanate, and shake for 5 min. Add 60 mL of methanol, complete to volume with water, and mix.

Sample stock solution: Transfer about 2.0 g of Trigonella foenum-graecum Seed, finely powdered and accurately weighed, to a centrifuge tube, add 8 mL of Diluent, heat in a water bath at 65º for 5 min, sonicated for 5 min and centrifuge. Transfer the supernatant into a 25-mL volumetric flask. Repeat the extraction two additional times and combine the extracts in the 25-mL volumetric flask. Complete to volume with Diluent, and mix.

Sample solution: Transfer 5.0 mL of Sample stock solution to a 50-mL volumetric flask, add 10 mL of Reagent and 500 µL of phenyl isothiocyanate, and shake for 5 min.  Add 30 mL of methanol, complete to volume with water, and mix. Before Injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first few mL of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 15-cm; 5-µm packing L1 (Similar to Zorbax Eclipse XD-C18, Luna C18(2) and Cosmosil C18-MS-II)

Flow rate: 1.5 mL/min

Injection volume: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0 for the 4-hydroxyisoleucine peak, Standard solution

Relative standard deviation: NMT 2.0%, determined from the 4-hydroxyisoleucine peak in repeated injections, Standard solution

Analysis

Samples: Standard solution and Sample solution

Using the chromatogram of Standard solution, identify the retention time of the peak corresponding to 4-hydroxyisoleucine in the Sample solution chromatogram.

Calculate the percentage of 4-hydroxyisoleucine in the portion of Trigonella foenum-graecum Seed taken:

 

Result = (rU/rS) × CS × (V/W) × d × 100

 

rU   = peak area of 4-hydroxyisoleucine from the Sample solution

rS   = peak area of 4-hydroxyisoleucine from Standard solution

CS  = concentration of 4-hydroxyisoleucine in Standard solution, (mg/mL)

  = volume of the Sample stock solution, (mL)

W   = weight of Trigonella foenum-graecum Seed taken to prepare the Sample stock solution, (mg)

d     = dilution factor to prepare the Sample solution from the Sample stock solution

Acceptance criteria: NLT 0.2%, on the dried basis

 

CONTAMINANTS

• Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 1.0 µg/g

Lead: NMT 10.0 µg/g

Mercury: NMT 1.0 µg/g

• Articles of Botanical Origin, General Method for Pesticide Residues Analysis <561>: 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

• Presence of Trigonelline

Standard solution A: 1.5 mg/mL of USP Trigonelline Hydrochloride RS in methanol

Standard solution B: 50 mg/mL of USP Trigonella foenum-graecum Seed Powdered Extract RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Sample solution: Use the Sample solution prepared in Identification B.

Chromatographic system

(See Chromatography <621>, Thin-Layer Chromatography.)

Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)

Application volume: 5 µL, as 8-mm bands

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

Temperature: 25°

Developing solvent system: Isopropyl alcohol, methanol, and water (4:1:4)

Developing distance: 6 cm

Derivatization reagent: Dragendorff’s reagent—suspend 1.7 g of bismuth oxynitrate and 20 g of (+)-tartaric acid in 40 mL of water. To the suspension add 40 mL of a 40% solution of potassium iodide in water (w/v), stir for 1 h, and filter. This stock solution may be kept for several days protected from light. Immediately before use, mix 5 mL of the stock solution with 15 mL of water.

Analysis

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

Apply the Samples as bands to a suitable HPTLC plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, dry, and examine under UV light at 254 nm. Derivatize with Derivatization reagent, dry, and examine under white light.

System suitability: Under UV light before derivatization, the chromatogram of Standard solution B exhibits, in the lower half, a quenching band at an RF similar to the trigonelline band in the chromatogram of Standard solution A.

Acceptance criteria: Under white light after derivatization, the chromatogram of Standard solution B exhibits, in the lower half, an orange-red band corresponding in RF and color to the trigonelline band in the chromatogram of Standard solution A.

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

• Articles of Botanical Origin, Alcohol-Soluble Extractives, Method 1 <561>: NLT 14.0%

• Articles of Botanical Origin, Water-Soluble Extractives, Method 1 <561>: NLT 28.0%

• Loss on Drying <731>

Analysis: Dry 1.0 g of Trigonella foenum-graecum Seed, finely powdered, at 105° for 2 h.

Acceptance criteria: NMT 5.0%

• Articles of Botanical Origin, Total Ash <561>

Analysis: 2.0 g of Trigonella foenum-graecum Seed, finely powdered

Acceptance criteria: NMT 5.0%

• Articles of Botanical Origin, Acid-Insoluble Ash <561>

Analysis: 6.0 g of Trigonella foenum-graecum Seed, finely powdered

Acceptance criteria: NMT 1.0%

 

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 of the plant contained in the article.

• USP Reference Standards <11>

USP 4-Hydroxyisoleucine RS

USP Trigonella foenum-graecum Seed Powdered Extract RS

USP Trigonelline Hydrochloride RS

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