Antrodia camphorata Fruiting Body Powder

Antrodia camphorata Fruiting Body Powder

Proposed For Comment Version 0.2

Antrodia camphorata Fruiting Body Powder

 


DEFINITION

The article consists of the dried fruiting bodies of Antrodia camphorata (M. Zang & C.H. Su) Sheng H. Wu, Ryvarden & T.T. Chang (Family Polyporaceae) reduced to a powder or very fine powder. It is also commonly known as Antrodia cinnamomea T.T. Chang & W.N. Chou. It contains: NLT 5% of triterpenoic acids, calculated as the sum of antcin A, antcin B, antcin C, antcin H, antcin K, dehydrosulfurenic acid, and dehydroeburicoic acid; and NLT 0.4% of antroquinonol; all calculated on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

Ganoderma camphoratum

 

CONSTITUENTS OF INTEREST

Triterpenoids: Antcin A, antcin B, antcin C, antcin H, antcin K, dehydrosulfurenic acid, and dehydroeburicoic acid

Methoxy-methylenedioxy aromatics: 1,4-Dimethoxy-2,3-methylenedioxy-5-methylbenzene

Polyacetylenes: Antrocamphin A and antrocamphin B

Cyclohexanone: Antroquinonol

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Red brown powder

• B. Thin-Layer Chrom​atography

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

Standard solution B: 0.3 mg /mL of USP Ergosterol RS in methanol

Standard solution C: 1.0 mg/mL of USP Antrodia camphorata Fruiting Body Dry Extract RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Sample solution: Sonicate about 100 mg of Antrodia camphorata Fruiting Body Powder in 1 mL of methanol for 10 min, centrifuge, and use the supernatant.

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, 10 µL of Standard solution C, and 5 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: Toluene, ethyl formate, and formic acid (5: 5: 0.2)

Developing distance: 7 cm

Derivatization reagent: 10% Sulfuric acid in methanol

Analysis

Samples: Standard solution A, Standard solution B, Standard solution C, 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 and heat for 5 min at 105°. Immediately examine under visible light and UV 365 nm.

System suitability: Under visible light, Standard solution C exhibits about five purple bands, one near the origin, three around the middle, and one near the solvent front. Under UV 365 nm, Standard solution C exhibits about seven bands with increasing RF: a pale yellow band near the origin, a pale blue band, a brown band, a blue band, a pale white band, and two light brown bands.

Acceptance criteria: Under visible light, the Sample solution exhibits a purple band corresponding in color and RF to ergosterol in Standard solution B, three purple bands above the ergosterol band, four purple bands below that reddish brown band corresponding to bands in Standard solution C, and one reddish brown band right below the ergosterol band. Under UV 365 nm, the Sample solution exhibits a pale brown band corresponding in color and RF to ergosterol in Standard solution C. About eight bands below the ergosterol band correspond in color and RF to the bands in Standard solution C with increasing RF from the origin: a yellow band, a bright blue band, a brown band, a bright yellow band, a pale yellow band, a brown band, a bright brown band, and a brown band. Three extra bands appear above the ergosterol band with increasing RF: a pale yellow band, a dark brown band, and a pink band.

• C. HPLC

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

Acceptance criteria: The chromatogram of the Sample solution exhibits peaks at the retention times corresponding to antcin A, antcin B, antcin C, antcin H, antcin K, 1,4-dimethoxy-2,3-methylenedioxy-5-methylbenzene, dehydrosulfurenic acid, and dehydroeburicoic acid in Standard solution B.

• D. HPLC

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

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

 

ASSAY

• Content of Triterpenoids

Solution A: 0.1% Formic acid in water

Solution B: Acetonitrile

Mobile phase: See Table 1.

 

Table 1

Time

(min)

Solution A

(%)

Solution B

(%)

0.0 70 30
7.5 50 50
10.8 40 60
13.8 35 65
16.8 32 68
18.5 25 75
30.6 15 85
35.6 0 100
35.7 70 30
38.0 70 30

 

Standard solution A: 0.1 mg/mL of USP Dexamethasone RS in methanol

Standard solution B: Transfer 10 mg of USP Antrodia camphorata Fruiting Body Dry Extract RS to a 10-mL volumetric flask, dilute with methanol, and sonicate. Before injection, pass through a PTFE filter of 0.2-μm pore size and discard the first portion of the filtrate.

Sample solution: Transfer 0.5 g of Antrodia camphorata Fruiting Body Powder to a 50-mL volumetric flask, dilute with methanol, and sonicate. Before injection, pass through a PTFE filter of 0.2-μm pore size and discard the first portion of the filtrate.

Chromatographic system

(See Chromatography <621> System Suitability.)

Mode: UHPLC

Detector: UV 248 nm

Column: 2.1-mm × 10-cm; 1.8-μm packing L1 (similar to ACQUITY UPLC HSS T3)

Column temperature: 40°

Flow rate: 0.3 mL/min

Injection volume: 5 μL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Chromatographic similarity: The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Antrodia camphorata Fruiting Body Dry Extract RS being used.

Resolution: NLT 1.0 between dexamethasone and antcin K peaks, Standard solution B

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

Relative standard deviation: NMT 2.0% determined from the dexamethasone 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 Antrodia camphorata Fruiting Body Dry Extract RS being used, identify the retention times of the peaks corresponding to antcin A, antcin B, antcin C, antcin H, antcin K, 1,4-dimethoxy-2,3-methylenedioxy -5-methylbenzene, dehydrosulfurenic acid, and dehydroeburicoic acid. The approximate relative retention times are provided in Table 2.

 

Table 2

Analyte

Approximate
Relative

Retention Times

Factor
Dexamethasone 1.00 1.00
(R,S)-Antcin K 1.29 0.34
1,4-Dimethoxy-2,3-methylenedioxy-5-methylbenzene 1.69 0.28
(R,S)-Antcin C 2.38 0.21
(R,S)-Antcin H 2.50 0.15
Dehydrosulfurenic acid 2.84 0.33
(R,S)-Antcin B 3.07 0.21
(R,S)-Antcin A 3.76 0.24
Dehydroeburicoic acid 5.87 0.47

 

Separately calculate the percentage of each of the triterpenes in the portion of Antrodia camphorata Fruiting Body Powder taken:

 

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

 

r = peak response of the analyte from the Sample solution

r = peak response of dexamethasone from Standard solution A

CS = concentration of dexamethasone in Standard solution A (mg/mL)

V   = volume of the Sample solution (mL)

= weight of Antrodia camphorata Fruiting Body Powder taken to prepare the Sample solution (mg)

F   = conversion factor for the analytes (see Table 2.)

[Note—Incorporate a factor in the calculation to account for the aliquot taken compared to the volume of the Sample solution.]

Add the percentages of antcin A, antcin B, antcin C, antcin H, antcin K, dehydrosulfurenic acid, and dehydroeburicoic acid.

Acceptance criteria: NLT 5% on the dried basis

• Content of Antroquinonol

Solution A: 0.3% Acetic acid in water

Solution B: Methanol

Mobile phase: See Table 3.

Table 3

Time

(min)

Solution A

(%)

Solution B

(%)

0 95 5
10 20 80
20 10 90
35 10 90
40 95 5

 

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

Standard solution B: Sonicate 1.0 mg/mL of USP Antrodia camphorata Fruiting Body Dry Extract RS in methanol for 10 min. Before injection, pass through a PTFE filter of 0.2-μm pore size and discard the first portion of the filtrate.

Sample solution: Transfer about 1 g of Antrodia camphorata Fruiting Body Powder to a flask and add 10.0 mL of methanol. Sonicate for 30 min and adjust to initial weight with methanol. Before injection, pass through a PTFE filter of 0.2-μm pore size and discard the first portion of the filtrate.

Chromatographic system

(See Chromatography <621> System Suitability.)

Mode: HPLC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; 5-μm packing L1 (similar to Hypersil C-18)

Flow rate: 1.0 mL/min

Injection volume: 20 μL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Chromatographic similarity: The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Antrodia camphorata Fruiting Body Dry Extract RS being used.

Resolution: NLT 1.0 between antroquinonol and the peak after, Standard solution B

Tailing factor: 1.1 for the antroquinonol peak, Standard solution A

Relative standard deviation: NMT 2% determined from the antroquinonol 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 Antrodia camphorata Fruiting Body Dry Extract RS being used, identify the retention time of the peak corresponding to antroquinonol.

Calculate the percentage of antroquinonol in the portion of Antrodia camphorata Fruiting Body Powder taken:

 

Result = (rU/rS) × CS × (V/W) × 100

 

rU   = peak response of the analyte from the Sample solution

rS   = peak response of antroquinonol from Standard solution A

C = concentration of antroquinonol in Standard solution A (mg/mL)

V    = volume of the Sample solution (mL)

W   = weight of Antrodia camphorata Fruiting Body Powder taken to prepare the Sample solution (mg)

[Note—Incorporate a factor in the calculation to account for the aliquot taken compared to the volume of the Sample solution.]

Acceptance criteria: NLT 0.4% 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 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, Test for Aflatoxins <561>: Meets the requirements

 

SPECIFIC TESTS

Articles of Botanical Origin, Alcohol-Soluble Extractives, Method 1 <561>: NLT 20%

Articles of Botanical Origin, Water-Soluble Extractives, Method 2 <561>: NLT 8.0%

Loss on Drying <731>

Sample: 1.0 g Antrodia camphorata Fruiting Body Powder

Analysis: Dry the Sample at 105° for 4 h.

Acceptance criteria: NMT 75%

Articles of Botanical Origin, Total Ash <561>: NMT 4%

 

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 Aflatoxins RS

USP Antrodia camphorata Fruiting Body Dry Extract RS

USP Antroquinonol RS

USP Dexamethasone RS

USP Ergosterol RS

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Proposed For Development Version 0.1