Cinnamomum cassia Twig Powder

Cinnamomum cassia Twig Powder

Proposed For Comment Version 0.2

Cinnamomum cassia Twig Powder

 


DEFINITION

The article consists of the dried twigs of Cinnamomum cassia (L.) J.Presl (Family Lauraceae), collected in spring or summer, reduced to a fine or very fine powder. It contains NLT 0.6% and NMT 2.0% of cinnamaldehyde (C9H8O), calculated on the anhydrous basis.

 

POTENTIAL CONFOUNDING MATERIALS

Cinnamomum burmannii (C. G. et Th. Nees) Bl., bark

Cinnamomum verum J. Presl, bark

Cinnamomum bejolghota (Buch.-Ham.) Sweet

Cinnamomum cassia (L.) J. Presl, bark

 

CONSTITUENTS OF INTEREST

Phenylpropanoids: Coumarin, cinnamic alcohol, cinnamic acid, 2-methoxycinnamic acid, cinnamaldehyde and 2-methoxycinnamaldehyde

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Reddish-brown

Microscopic:  Stone cells subsquare or subrounded, 30–64 μm in diameter with thickened walls, some with a very thin wall at one side. Phloem fibers mostly in bundles or scattered singly, colorless or brown, fusiform, some margin serrate, 12–40 μm in diameter, with heavily thickened walls, lignified, pit canals indistinct. Oil cells subrounded or elliptical, 41–104 μm in diameter. Xylary fibers numerous, usually in bundles, pits oblique or crossed. Cork cells yellowish-brown, polygonal in surface view, containing reddish-brown content. Vessels mainly with bordered pits, up to 76 μm in diameter.

• B. Thin-Layer Chromatography

Standard solution A: 0.5 mg/mL of USP Cinnamaldehyde RS in methanol

Standard solution B: 100 mg/mL of USP Cinnamomum cassia Twig Dry Extract RS in toluene, sonicate for 10 min, centrifuge, and use the supernatant.

Sample solution: Sonicate 2.0 g of Cinnamomum cassia Twig Powder in 10 mL of methanol for 10 min, centrifuge or filter, and transfer the extract to a round bottom flask. Wash the remaining plant material with 5 mL of methanol and combine the washing with the extract. Evaporate the solution under reduced pressure to dryness. Dissolve the residue in 2 mL of toluene, sonicate for about 2 min, centrifuge, and use the supernatant.

Chromatographic system

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

Adsorbent: Chromatographic silica gel F254 mixture with an average particle size of 5 µm (HPTLC plates)

Application volume: 6 μL, as 8-mm bands

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

Developing solvent system: Toluene and ethyl acetate (95:5)

Developing distance: 7 cm

Derivatization reagent: Methanol, acetic acid, sulfuric acid, and ρ-anisaldehyde (170:20:10:1). [Note—Prepare fresh. Slowly add sulfuric acid to ice-cold methanol, followed by acetic acid and ρ-anisaldehyde.]

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 (20 min with filter paper), remove the plate from the chamber, and air-dry. Examine under UV light at 254 nm. Then dip (time 0, speed 5) the plate with the Derivatization reagent, heat at 100° for 2 min, and examine under UV light at 366 nm. 

System suitability: Under UV light at 254 nm, Standard solution B exhibits, in the middle-third section, an intense quenching band corresponding in RFto the band for cinnamaldehyde in Standard solution A. In the lower-third section, Standard solution B exhibits one weak quenching band in the upper part due to coumarin, one quenching band close to the starting position due to cinnamic acid, and one quenching band between coumarin and cinnamic acid due to cinnamic alcohol. After derivatization, under UV light at 366 nm, Standard solution B exhibits a band corresponding in RFand color to the band for cinnamaldehyde in Standard solution A, and one yellow band immediately below the band of cinnamaldehyde. 

Acceptance criteria: Under UV light at 254 nm, the Sample solution exhibits, in the middle-third section, an intense quenching band corresponding in RFto the band for cinnamaldehyde in Standard solution A. The Sample solution exhibits additional bands corresponding to similar bands in Standard solution B; these include one weak quenching band in the upper part of the lower-third section due to coumarin, one quenching band close to the starting position due to cinnamic acid, and one quenching band between the bands of coumarin and cinnamic acid. After derivatization, under UV light at 366 nm, the Sample solution exhibits a band corresponding in RFand color to the band for cinnamaldehyde in Standard solutions A and B, and one yellow band immediately below the band of cinnamaldehyde. There is no red band immediately above the cinnamaldehyde band (distinguished from Cinnamomum verum).

• C. HPLC

Analysis: Proceed as directed in the Assay for Content of Cinnamaldehyde.

Acceptance criteria: The Sample solution exhibits an intense peak with a retention time corresponding to cinnamaldehyde in Standard solution A; and exhibits peaks related to coumarin and cinnamic alcohol (co-eluted), cinnamic acid, and 2'-methoxycinnamaldehyde at retention times corresponding to the same phenylpropanoids in Standard solution B and the reference chromatogram provided with the lot of USP Cinnamomum cassia Twig Dry Extract RS being used. The peak area ratios for each peak versus the cinnamaldehyde peak are listed in Table 1.

 

ASSAY

• Content of Cinnamaldehyde 

Solution A: 0.01% Phosphoric acid in water

Solution B: Acetonitrile

Mobile phase: Solution A and Solution B (65:35). [Note—Protect from light and proceed under low actinic light. The Standard solutions and Sample solution are stable for 24 h at room temperature.]

Standard solution A: 0.06 mg/mL of USP Cinnamaldehyde RS in methanol

Standard solution B: 1 mg/mL of USP Cinnamomum cassia Twig Dry Extract RS in methanol, sonicate, centrifuge, and pass through a membrane filter of 0.45-μm or finer pore size.

Sample solution: Accurately transfer about 250 mg of Cinnamaldehyde cassia Twig Powder into a suitable flask and accurately add 25 mL of methanol. Sonicate for 30 min and filter into a 50-mL volumetric flask. Rinse the flask and the residue left in the flask with 10 mL of methanol, and wash the residue and paper on the filter using the rinsing. Repeat the rinse procedure one more time. Continue to wash the paper on the filter with 4 mL of methanol, dilute with methanol to volume, and mix. Before injection, pass through a membrane filter of 0.45-μm or finer pore size and discard the first portion of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 290 nm

Column: 4.6-mm × 10-cm; 3.5-μm packing L1 (similar to Agilent Zorbax SB C18)

Column temperature: 30°

Flow rate: 1.2 mL/min

Injection volume: 5 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 Cinnamomum cassia Twig Dry Extract RS being used.

Resolution: NLT 1.5 between the peak of coumarin and cinnamic alcohol (co-eluted) and the peak of cinnamic acid, Standard solution B

Tailing factor: NMT 2.0 for cinnamaldehyde peak, Standard solution A

Relative standard deviation: NMT 2.0% for cinnamaldehyde, 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 Cinnamomum cassia Twig Dry Extract RS being used, identify the retention times of the peaks corresponding to coumarin and cinnamic alcohol (co-eluted), cinnamic acid, cinnamaldehyde, and 2'-methoxycinnamaldehyde in the Sample solution. [Note—The approximate relative retention times for each peak are provided in Table 1.]

 

 Table 1

Analyte

Approximate Relative
Retention Times

Peak Area Ratio

Coumarin and cinnamic alcohol

0.58

1.0–3.0

Cinnamic acid

0.63

2.0–10

Cinnamaldehyde

1.00

100

2'-methoxycinnamaldehyde

1.35

6.0–13

 

Calculate the percentage of cinnamaldehyde in the portion of Cinnamomum cassia Twig Powder taken:

 

Result = (rU/rS) × CS × (V/W) × 100

 

rU   = peak area of cinnamaldehyde from the Sample solution

rS   = peak area of cinnamaldehyde from Standard solution A

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

V    = volume of the Sample solution (mL)

W   = weight of Cinnamomum cassia Twig Powder taken to prepare the Sample solution (mg)

Acceptance criteria: 0.6%–2.0% on the anhydrous basis

 

CONTAMINANTS

Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 0.3 µg/g

Lead: NMT 5.0 µg/g

Mercury: NMT 0.2 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

Articles of Botanical Origin, Aflatoxins <561>: Meets the requirements

 

SPECIFIC TESTS

Articles of Botanical Origin, Alcohol-Soluble Extractives, Method 1 <561>: NLT 6.0%

Water Determination, Method II <921>: NMT 12.0%

Articles of Botanical Origin, Total Ash <561>: NMT 3.0%

Articles of Botanical Origin, Acid-Insoluble Ash <561>: NMT 0.5%

 

ADDITIONAL REQUIREMENTS

• 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 the part(s) of the plant contained in the article.

USP Reference Standards <11>

USP Aflatoxins RS

USP Cinnamaldehyde RS

USP Cinnamomum cassia Twig Dry Extract RS

 

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Other Versions

Final Authorized Version 1.0
Proposed For Comment Version 0.3
Proposed For Development Version 0.1