Citrus reticulata Peel

Citrus reticulata Peel

Final Authorized Version 1.0

Citrus reticulata Peel

 


 

DEFINITION

The article consists of the dried exocarp and mesocarp of the ripe fruit of Citrus reticulata Blanco (Family Rutaceae), partly freed from the white spongy tissue of the mesocarp. It contains NLT 5.0% of total dihydroflavone glycosides, calculated as the sum of narirutin (C27H32O14), hesperidin (C28H34O15), and didymin (C28H34O14) on the anhydrous basis; NLT 0.1% of total polymethoxylated flavones, calculated as the sum of nobiletin (C21H22O8), 3,5,6,7,8,3',4'-heptamethoxyflavone (C22H24O9), and tangeretin (C20H20O7) on the anhydrous basis.

 

SYNONYMS

Citrus chrysocarpa Lush

Citrus crenatifolia Lush

Citrus daoxianensis S. W. He & G. F. Liu

Citrus deliciosa Ten

Citrus depressa Hayata

Citrus erythrosa Yu. Tanaka

Citrus himekitsu Yu. Tanaka

Citrus otachihana Yu. Tanaka

Citrus papillaris Blanco

Citrus vangasay Bojer

 

POTENTIAL CONFOUNDING MATERIALS

Dried ripe fruit of Citrus wilsoni Tanaka

Dried unripe peel of Citrus maxima (Burm.) Merr. (C. grandis (L.) Osbeck)

 

SELECTED COMMON NAMES

Chinese: 陈皮, 陳皮 (Chen Pi)

English: Tangerine peel

French: Écorce de mandarine

Japanese: ちんぴ

Spanish: Cáscara de mandarina

 

CONSTITUENTS OF INTEREST

Dihydroflavone glycoside: Narirutin, hesperidin, and didymin

Polymethoxylated flavone: Nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Usually in several lobes connected at the end, or in irregular slices with a thickness of 1–4 mm. External surface orange-red or reddish brown with fine wrinkles and sunken dotted oil cavity; interior surface pale yellowish white, rough, with yellowish-white or yellowish-brown vein-like vascular bundles. Texture hard and fragile.

Microscopic

Transverse section: Epidermal cells subsquare, covered with cuticle, sometimes stomata visible. Parenchymatous cells with thickened walls containing calcium oxalate prisms and hesperidin crystals in spheroid or amorphous mass, sometimes vessels visible. Inner cells subround, loosely arranged with uneven thickened walls. Oil cavities consisting of 1–2 layers of cells, ovate or elliptical, arranged irregularly and mainly scattered at the outer side of mesocarp.

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

Standard solution A: 1.0 mg/mL of USP Hesperidin RS in methanol. Sonicate to dissolve.

Standard solution B: 50 mg/mL of USP Citrus reticulata Pericarp Dry Extract RS in methanol. Sonicate for 20 min, centrifuge, and use the supernatant.

Sample solution: 500 mg of Citrus reticulata Peel, finely powdered, in 5 mL of methanol. Sonicate for 20 min, centrifuge, and use the supernatant.

Chromatographic system

Adsorbent: Chromatographic silica gel F254 mixture

Application volume: 10 µL for Standard solution A; 5 µL each for Standard solution B and Sample solution, 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: Ethyl acetate, formic acid, and water (100:15:13)

Derivatization reagent A: 10 mg/mL of 2-aminoethyl diphenylboriate in methanol

Derivatization reagent B: 50 mg/mL of polyethylene glycol 4000 in alcohol

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, remove the plate from the chamber, and dry the plate at 100° for 3 min. Treat the plate with Derivatization reagent A and dry for 5 min with a stream of cool air. Immediately, treat the plate with Derivatization reagent B, dry for 5 min with a stream of cool air, and examine under UV light at 366 nm.

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements: Standard solution A exhibits a yellowish-brown band due to hesperidin in the lower-half section. Standard solution B exhibits a band corresponding in RF and color to the band due to hesperidin in Standard solution A, a yellow band below hesperidin, a blue band below the yellow band, another blue band above hesperidin, and a light yellowish-brown band between hesperidin and the blue band above. In the upper-third section, Standard solution B exhibits a bright blue band due to the coelution of nobiletin with some other components, and a faint green band above the bright blue band.

Acceptance criteria: The Sample solution exhibits a yellowish-brown band corresponding in RF and color to the band due to hesperidin in Standard solution A and Standard solution B, a yellow band below hesperidin, a blue band below the yellow band, another blue band above hesperidin, and a light yellowish-brown band between hesperidin and the blue band above corresponding in RF and color to the same bands in Standard solution B. In the upper-third section, the Sample solution exhibits a bright blue band and a faint green band above the bright blue band corresponding in RF and color to the same bands in Standard solution B. In the lower-third section, the Sample solution exhibits a couple of yellow bands close to the starting position and a couple of faint bands above the yellow bands corresponding in RF  and color to the same bands in Standard solution B.

• C. HPLC

Analysis: Proceed as directed in the Assay for Content of Dihydroflavone Glycosides and Polymethoxylated Flavones.

Acceptance criteria: The Sample solution exhibits the most intense peak at the retention time corresponding to hesperidin in Standard solution A and peaks due to narirutin, didymin, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin at retention times corresponding to the same constituents in Standard solution B. No other peak between narirutin and tangeretin is more intense than the peak corresponding to didymin (a distinction from other Citrus species; Citrus maxima Peel and Citrus wilsonii Fruit show a principal peak for naringin). The content ratios of narirutin and didymin relative to hesperidin are within the ranges listed in Table 2.

 

ASSAY

• Content of Dihydroflavone Glycosides and Polymethoxylated Flavones

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

85

15

8

81

19

10

81

19

17

60

40

28

56

44

[Note—The Standard solutions and the Sample solution are stable for 24 h at room temperature.]

Standard solution A: 0.40 mg/mL of USP Hesperidin RS in methanol

Standard solution B: 0.05 mg/mL of USP Nobiletin RS in methanol

Standard solution C: 5 mg/mL of USP Citrus reticulata Pericarp Dry Extract RS in methanol. Sonicate for 15 min, centrifuge, and pass through a suitable membrane filter of 0.22-μm pore size.

Sample solution: Accurately transfer about 100 mg of finely powdered Citrus reticulata Peel to a suitable flask, accurately add 10.0 mL of methanol, and close tightly. Weigh the filled flask accurately, and sonicate for 30 min. Cool to room temperature and adjust to the initial weight by adding methanol if needed. Before injection, pass through a suitable membrane filter of 0.22-μm pore size and discard the first portion of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 283 nm (0–17 min) and 330 nm (17–28 min)

Column: 4.6-mm × 5-cm; 1.8-μm packing L1 (similar to Agilent Zorbax SB C18)

Column temperature: 25°

Flow rate: 0.7 mL/min

Injection volume: 2 µL

System suitability

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

Suitability requirements

Resolution: NLT 1.5 between the peak of hesperidin and the small peak before it, Standard solution C

Tailing factor: NMT 1.5 for the hesperidin and nobiletin peaks, Standard solution A and Standard solution B

Relative standard deviation: NMT 2.0% for the hesperidin and nobiletin peaks, Standard solution A and Standard solution B

Chromatogram similarity: The chromatogram of Standard solution C is similar to the reference chromatogram provided with the lot of USP Citrus reticulata Pericarp Dry Extract RS being used.

Analysis

   For dihydroflavone glycosides

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

Using the chromatograms of Standard solution A, Standard solution C, and the reference chromatogram provided with the lot of USP Citrus reticulata Pericarp Dry Extract RS being used, identify the peaks corresponding to narirutin, hesperidin, and didymin in the Sample solution. [Note—See Table 2 for relative retention times.]

 

Table 2

Analyte

Approximate Relative
Retention Time

Conversion Factor

Content Ratio Relative to Hesperidin

Narirutin

0.8

1.17

0.1–0.3

Naringin 0.9 <0.02

Hesperidin

1.0

1.0

1.0

Didymin

1.7

1.0

0.02–0.06

 

Separately calculate the percentage of narirutin, hesperidin, and didymin in the portion of Citrus reticulata Peel taken:

 

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

 

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

rS   = peak area of hesperidin from Standard solution A

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

   = volume of the Sample solution (mL)

W   = weight of Citrus reticulata Peel taken to prepare the Sample solution (mg)

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

 

Calculate the content of total dihydroflavone glycosides as the sum of the percentages of narirutin, hesperidin, and didymin.

 

For polymethoxylated flavones

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

Using the chromatograms of Standard solution B, Standard solution C, and the reference chromatogram provided with the lot of USP Citrus reticulata Pericarp Dry Extract RS being used, identify the peaks corresponding to nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin in the Sample solution. [Note—See Table 3 for relative retention times.]

 

Table 3

Analyte

Approximate Relative
Retention Time

Conversion Factor

Nobiletin

1.00

1.00

3,5,6,7,8,3',4'-Heptamethoxyflavone

1.06

1.32

Tangeretin

1.12

0.88

 

Separately calculate the percentage of nobiletin, 3,5,6,7,8,3',4'- heptamethoxyflavone, and tangeretin in the portion of Citrus reticulata Peel taken:

 

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

 

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

rS     = peak area of nobiletin from Standard solution B

C = concentration of USP Nobiletin RS in Standard solution B (mg/mL)

V     = volume of the Sample solution (mL)

W    = weight of Citrus reticulata Peel taken to prepare the Sample solution (mg)

F     = conversion factor for the analyte (see Table 3)

 

Calculate the content of total polymethoxylated flavones as the sum of the percentages of nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin.

Acceptance criteria

Total dihydroflavone glycosides: NLT 5.0% on the anhydrous basis

Total polymethoxylated flavones: NLT 0.1% 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

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

 

SPECIFIC TESTS

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

• Articles of Botanical Origin <561>, Methods of Analysis, Alcohol-Soluble Extractives, Method 1: NLT 15.0%

• Articles of Botanical Origin <561>, Methods of Analysis, Water-Soluble Extractives, Method 2: NLT 30.0%

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

• Articles of Botanical Origin <561>, Methods of Analysis, Total Ash: NMT 5.0%

• 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 Citrus reticulata Pericarp Dry Extract RS

USP Hesperidin RS

USP Nobiletin RS

Other Versions

Proposed For Comment Version 0.2
Proposed For Development Version 0.1