Cinnamomum cassia Twig Powder

Cinnamomum cassia Twig Powder

Final Authorized Version 1.0

Cinnamomum cassia Twig Powder

 


DEFINITION

The article consists of the dried twigs of Cinnamomum cassia (L.) J. Presl with a synonym Cinnamomum aromatium Nees (Family Lauraceae), collected in spring or summer, reduced to a fine or very fine powder. It contains NLT 0.8% of total phenylpropanoids calculated as the sum of cinnamyl alcohol (C9H10O), cinnamic acid (C9H8O2), 2-methoxycinnamic acid (C10H10O3), cinnamaldehyde (C9H8O), and 2-methoxycinnamaldehyde (C10H10O2) on the anhydrous basis.

 

POTENTIAL CONFOUNDING MATERIALS

Cinnamomum bejolghota (Buch.-Ham.) Sweet, bark

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

Cinnamomum cassia (L.) J.Presl, bark

Cinnamomum verum J.Presl, bark

 

CONSTITUENTS OF INTEREST

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

Coumarin

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Reddish-brown powder

Microscopic:  Stone cells sub-squared or sub-rounded, 30–65 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, 10–40 μm in diameter, with heavily thickened walls, lignified, pit canals indistinct. Oil cells are sub-rounded or elliptical, 40–105 μm in diameter. Xylary fibers are numerous, usually in bundles, pits oblique or crossed. Cork cells yellowish-brown, polygonal in surface view, containing reddish-brown content. Vessels are mainly with bordered pits, up to 80 μm in diameter.

• B. HPTLC For Articles of Botanical Origin <203>

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

Standard solution B: USP Cinnamomum cassia Twig Powder RS in methanol (1 in 5). Sonicate for 10 min, centrifuge or filter, and evaporate the solvent. Suspend the residue in a volume of toluene equivalent to 1/5 the volume of methanol used for the extraction. Sonicate for about 2 min, centrifuge or filter, and use the supernatant or filtrate.

Sample solution: 2 g of Cinnamomum cassia Twig Powder in 10 mL of methanol. Sonicate for 10 min, centrifuge or filter, and transfer the extract to a round-bottom flask. Evaporate the solution under reduced pressure to dryness. Dissolve the residue in 2 mL of toluene, sonicate for about 2 min, centrifuge or filter, and use the supernatant or filtrate.

Chromatographic system

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

Application volume: 6 μL, as 8-mm bands

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

Temperature: About 25°

Developing solvent system: Toluene and ethyl acetate (19:1)

Developing distance: 6 cm

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

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 nm. Then treat the plate with Derivatization reagent, heat at 100° for 2 min, and examine under UV light at 366 nm.

System suitability: Prior to derivatization, under UV light at 254 nm, Standard solution A exhibits a quenching band due to cinnamaldehyde in the middle third of the chromatogram, and a quenching band due to coumarin in the upper part of the lower third. Upon derivatization, under UV light at 366 nm, the cinnamaldehyde band is seen as light-blue, while the coumarin band assumes a deeper blue color. Under UV light at 254 nm, Standard solution B exhibits, in the middle-third section, an intense quenching band corresponding in RF  to the cinnamaldehyde band in Standard solution A. In the upper part of the lower-third section, Standard solution B exhibits a weak quenching band due to coumarin, a quenching band close to the starting position due to cinnamic acid, and a quenching band between coumarin and cinnamic acid due to cinnamyl alcohol. After derivatization, under UV light at 366 nm, Standard solution B exhibits a band corresponding in Rand color to the cinnamaldehyde band in Standard solution A, and a yellow band immediately below the cinnamaldehyde band.

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

• C. HPLC

Analysis: Proceed as directed in the Assay for Content of Total Phenylpropanoids and Coumarin.

Acceptance criteria: The Sample solution exhibits the most intense peak at a retention time corresponding to cinnamaldehyde in Standard solution B; a peak with a retention time corresponding to cinnamic acid in Standard solution A; and peaks due to coumarin, cinnamyl alcohol, 2-methoxycinnamic acid, and 2-methoxycinnamaldehyde at retention times corresponding to the same constituents in Standard solution B. The content ratios of these constituents relative to cinnamic acid are within the typical ratio ranges listed in Table 2. The content of cinnamaldehyde is NLT 65% of the content of total phenylpropanoids.

 

ASSAY

• Content of Total Phenylpropanoids and Coumarin

Solution A: 0.05% phosphoric acid in water

Solution B: Acetonitrile

Mobile phase: See Table 1.

Table 1 

Time
(min)

Solution A
(%)

Solution B
(%)

0

75

25

1

75

25

21

62

38

30

60

40

35

60

40

 

[Note—Protect from light and proceed under low actinic light. Standard solution A, Standard solution B, and Sample solution are stable for 24 h at room temperature.]

Solvent: Methanol and water (7:3)

Standard solution A: 0.05 mg/mL of USP Cinnamic Acid RS in methanol

Standard solution B: Transfer about 250 mg of USP Cinnamomum cassia Twig Powder RS to a 50-mL round-bottom centrifuge tube, and add 25 mL of Solvent. Sonicate for 30 min, cool, and centrifuge. Before injection, pass through a membrane filter of 0.45-µm or finer pore size.

Sample solution: Accurately transfer about 500 mg of Cinnamomum cassia Twig Powder to a 50-mL round-bottom centrifuge tube, and add 25 mL of Solvent. Sonicate for 30 min (250W, 33 kHz), cool, centrifuge, and transfer the supernatant to a 50-mL volumetric flask. Repeat the extraction one more time by adding 15 mL of Solvent, and transfer the supernatant to the same 50-mL volumetric flask, dilute with Solvent 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 265 nm

Column: 4.6-mm × 25-cm; 5-μm packing L1 [similar to Phenomenex Luna C18 (2) 100Å]

Column temperature: 25°

Flow rate: 1.0 mL/min

Injection volume: 10 µL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Resolution: NLT 1.5 between the cinnamyl alcohol and cinnamic acid peaks, and the coumarin and cinnamyl alcohol peaks, Standard solution B

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

Relative standard deviation: NMT 2.0% for the cinnamic acid peak, Standard solution A

Chromatogram similarity: The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Cinnamomum cassia Twig Powder RS being used.

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 Powder RS being used, identify the retention times of the peaks corresponding to coumarin, cinnamyl alcohol, cinnamic acid, 2-methoxycinnamic acid, cinnamaldehyde, and 2-methoxycinnamaldehyde in the Sample solution. [Note—See Table 2 for relative retention times.]

 

 Table 2

Analyte

Approximate
Relative
Retention Time

Conversion
Factor

Typical Content
Range

(%)

Typical Ratio
Relative to
Cinnamic Acid

Coumarin

0.8

2.10

0.02–0.15

0.2–1.3

Cinnamyl alcohol

0.9

2.12

0.003–0.05

0.02–0.6

Cinnamic acid

1.0

1.00

0.04–0.16

1.0

2-Methoxycinnamic acid

1.1

1.82

0.002–0.02

0.04–0.16

Cinnamaldehyde

1.3

1.47

0.6–2.0

6–30

2-Methoxycinnamaldehyde

1.5

2.69

0.08–0.5

0.5–6.0

 

Separately calculate the percentages of coumarin, cinnamyl alcohol, cinnamic acid, 2-methoxycinnamic acid, cinnamaldehyde, and 2-methoxycinnamaldehyde in the portion of Cinnamomum cassia Twig Powder taken:

 

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

 

rU   = peak area of the relevant analyte from the Sample solution

rS   = peak area of cinnamic acid from Standard solution A

CS  = concentration of USP Cinnamic Acid 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)

F    = conversion factor for the analyte (see Table 2)

 

Calculate the content of total phenylpropanoids as the sum of cinnamyl alcohol, cinnamic acid, 2-methoxycinnamic acid, cinnamaldehyde, and 2-methoxycinnamaldehyde.

Acceptance criteria: NLT 0.8% of total phenylpropanoids on the anhydrous basis

 

CONTAMINANTS

Articles of Botanical Origin <561>, Limits of Elemental Impurities: Meets the requirements

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 count 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>, Alcohol-Soluble Extractives, Method 1: NLT 6.0%

Water Determination <921>, Method II: NMT 15.0%

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

Articles of Botanical Origin <561>, Methods of Analysis, Acid-Insoluble Ash: NMT 1.0%

 

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 following the official name of the plant contained in the article.

USP Reference Standards <11>

USP Cinnamaldehyde RS

USP Cinnamic Acid RS

USP Cinnamomum cassia Twig Powder RS

 

Other Versions

Proposed For Comment Version 0.3
Proposed For Comment Version 0.2
Proposed For Development Version 0.1