Antrodia camphorata Fruiting Body

Antrodia camphorata Fruiting Body

Proposed For Comment Version 0.2

Antrodia camphorata Fruiting Body

 


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). 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 on the dried basis.

 

SYNONYMS

Antrodia cinnamomea T.T. Chang & W.N. Chou

Taiwanofungus camphoratus (M. Zang & C.H. Su) Sheng H. Wu, Z.H. Yu, Y.C. Dai & C.H. Su

 

POTENTIAL CONFOUNDING MATERIALS

Ganoderma camphoratum

 

SELECTED COMMON NAMES

Chinese: 牛樟芝 (niu-chang-ku)

 

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

Cyclohexenone: Antroquinonol

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Fruiting body is 10–15 cm broad, 0.5–1 cm thick, red to light-cinnamon, resupinate, effused-reflexed to pileate, cylindrical basidiospores, and weakly amyloid skeletal hyphae; effused-reflexed to more or less triquetrous, and elongated to semicircular. This basidiocarp is tightly attached to the lateral base and may form subpendant and irregular; sterile in margin, corky to woody. Upper surface of the basidiocarp at first colors with orange-red and organizes orange-brown to light-cinnamon resinous layer. Further, this surface character persists over the younger marginal areas, becoming brown or blackish, glabrous, concentrically-zoned, and sulcate; the margin of the surface is obtuse, deflexed, and undulate. Pore is round to angular, 4–6 mm; pore surface is orange-red, but orange-brown to light-cinnamon in young specimens, and cream-colored to buff in old specimens. Context is lacking or very thin, and concolorous with the pore surface; tube is up to 40 mm long, not stratified, and concolorous with the pore surface.

Microscopic: The hyphae are generative with 2.0–3.5 µm clamp connections, and hyaline to light-brown skeletal hyphae are up to 4.5 µm wide with weak amyloid. Basidia, 12–14 x 3–5 µm, are clavate and 4-sterigmate with a basal clamp. Basidiospores, 3.5–5.0 x 1.5–2.0 µm, are cylindrical, hyaline, smooth, and sometimes slightly bent.

• B. Thin-Layer Chromatography

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

Standard solution B: 0.3 mg /mL 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, finely powdered, 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, the chromatogram of Standard solution C exhibits about five purple bands, one near origin, three around the middle of the chromatogram, and one near solvent front. Under UV 365 nm, the chromatogram of Standard solution C exhibits about seven bands with increasing RF: a pale yellow band near 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 chromatogram of Sample solution exhibits a purple band corresponding in color and RF to ergosterol in the chromatogram of Standard solution B; three purple bands above the ergosterol band and one reddish brown band right below ergosterol band; four purple bands below that reddish brown band correspond to bands in the chromatogram of Standard solution C; one reddish brown band right below ergosterol band. Under UV 365 nm, the chromatogram of Sample solution exhibits a pale brown band corresponding in color and RF to ergosterol in the chromatogram of Standard solution C. About eight bands below the ergosterol band corresponding in color and RF to the bands in the chromatogram of 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 ergosterol bands with increasing RF; a pale yellow, a dark brown, and a pink bands.

• C. HPLC

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

Acceptance criteria: The Sample solution chromatogram 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, dehydroeburicoic acid in the chromatogram of Standard solution B.

• D. HPLC

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

Acceptance criteria: The Sample solution chromatogram exhibits peaks at the retention times corresponding to antroquinonol in the chromatogram of 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 having a 0.2-μm pore size and discard the first portion of the filtrate.

Sample solution: Transfer 0.5 g of Antrodia camphorata Fruiting Body, finely powdered and accurately weighed, to a 50-mL volumetric flask, dilute with methanol, and sonicate. Before injection, pass through a PTFE filter having a 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 of the different peaks 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 taken:

 

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

 

rU  = peak response of the analyte from the Sample solution

rS  = peak responses of dexamethasone from Standard solution A

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

V   = volume of the Sample solution (mL)

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

F   = conversion factors 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 having a 0.2-μm pore size and discard the first portion of the filtrate.

Sample solution: Transfer about 1 g of Antrodia camphorata Fruiting Body, finely powdered and accurately weighed, 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 having a 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 times of the peak corresponding to antroquinonol.

Separately calculate the percentage of antroquinonol in the portion of Antrodia camphorata Fruiting Body taken:

 

Result = (rU/rS) × CS × (V/W) × 100

 

rU  = peak response of the analyte from the Sample solution

rS  = peak responses of antroquinonol from Standard solution A

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

V   = volume of the Sample solution (mL)

W  = weight of Antrodia camphorata Fruiting Body 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.]

Calculate the content of antroquinonol.

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, finely powdered

Analysis: Dry 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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1 comment

Hsin-Wen H.posted Tuesday, March 31, 2015 - 1:17am
To whom it may concern: This is Taiwan Niu-Chang-Chih Industry Association (TNIA) and the members of the Association include scholars and scientists from Taiwan’s universities and academic institutes and companies interesting in Antrodia cinnamomea industry in Taiwan. We recently noticed the information that the Herbal Medicines Compendium in USP web site (https://hmc.usp.org) proposed the reference standard for A. camphorate Fruiting Body/Fruiting Body Dry Extract, we therefore would like to submit our proposal as follows for your consideration: 1. According to the standard proposed by the Bureau of Standards, Metrology & Inspection (BSMI), the Ministry of Economic Affairs (MOEA), Taiwan, fruiting body of A. cinnamomea can be identified by appearance, fragrancy, flavor, and microscopic observation. Water content of the fresh or dry mushroom should be below 80% or 10%, respectively. The 16S Internal Transcirbed Spacer (ITS) sequence of the strain should have more than 98% identity as compared with the reference standard strain of BCRC 35398. Ethanol extraction rate is higher than 25% of dry fruiting body weight. In addition, antcin A, antcin B, antcin C, antcin H, antcin K, 4,7-dimethoxy-5-methyl-1, 3-benzodioxole, dehydrosulfurenic acid, dehydroeburicoic acid are 8 Index components in the ethanol extract of A. cinnamomea that should be detected by HPLC analysis (below). HPLC Analysis Conditions: 1. Sample conc.: 10 mg/mL in methanol 2. Injection volume: 2.0 μL 3. Flow rate: 0.25 mL/min. 4. UV detector: 210 nm or 254 nm 5. Column temp.: 30o C 6. Mobil phase solution: Solution A: 0.1% Formic acid Solution B: Acetonitrile Time (min.) Solution A (%) Solution B (%) 0 70 30 40 50 50 60 50 50 80 100 0 120 100 0 2. The 8 Index components in BSMI’s proposed standard, cannot be analyzed quantitatively by this HPLC method. However, there is one analytical method according to a paper published in J. Agric. Food Chem. (2011 59:7626-35.) that can determine both qualitatively and quantitatively the 13 representative metabolites in the fruiting body of A. cinnamomea. Another advantage of these 13 representative metabolites is that it can distinguish the origin of the sporocarp of A. cinnamomea grown from the original host (Cinnamomum kanehirai) and various other tree species. 3. Please kindly consider Taiwan BSMI’s proposal for the identification of A. camphorate fruiting body, and adopt the study published in J. Agric. Food Chem. to clarify the origin of the fruiting body of A. camphorata. Sincerely Yours, Taiwan Niu-Chang-Chih Industry Association

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