Lonicera japonica Flower Powder. Proposed For Development Version 0.1. Published on Herbal Medicines Compendium (
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1 Published on Herbal Medicines Compendium ( Lonicera japonica Flower Powder Proposed For Development Version 0.1 Lonicera japonica Flower Powder DEFINITION The article consists of the dried flower buds or in early opening flowers of Lonicera japonica Thunb. (Family Caprifoliaceae), collected before flowering in early summer, reduced to a fine or very fine powder. It contains NLT 3.8% of caffeoylquinic acids, calculated as the sum of chlorogenic acid, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid; NLT 0.80% of iridoids, calculated as the sum of sweroside, secoxyloganin and centauroside, all calculated on the dried basis. POTENTIAL CONFOUNDING MATERIALS Lonicera macranthoides Flower powder Lonicera hypoglauca Flower powder Lonicera confusa Flower powder Lonicera fulvotomentosa Flower powder CONSTITUENTS OF INTEREST Iridoids: Secoxyloganin, sweroside, centauroside Caffeoylquinic acids: Neochlorogenic acid, Chlorogenic acid, Cryptochlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-Ocaffeoylquinic acid and 4,5-di-O-caffeoylquinic acid Flavone: Luteolin-7-O-glucoside, Rutin IDENTIFICATION A. BOTANICAL CHARACTERISTICS Macroscopic: Yellowish-white to yellowish-green. Microscopic: Glandular hairs are numerous. The head of glandular hairs are multicellular in subround or slightly oblate, μm in diameter, sometimes up to 108 μm. The stalks of glandular hairs are unicellular or multicellular, 2 70 μm long, sometimes up to 700 μm. Non-glandular hairs occur in two types, one with thick walls, unicellular, μm long, μm in diameter, with fine verrucae on the surface, some have corneous spiral; another type with thin walls, slender, curved or shrinkage, with fine verrucae on the surface. Clusters of calcium oxalate are 6 45 μm in diameter. Pollen grains are spherical, with 3 germinal pores, μm in diameter. B. THIN-LAYER CHROMATOGRAPHY Standard solution A: 1.0 mg/ml of USP chlorogenic acid RS and 0.5 mg/ml USP luteolin-7-o-glucoside RS in methanol Standard solution B: 5.0 mg/ml of USP Lonicera japonica Flower Dry Extract RS in methanol, sonicate for 10 min and filter. Sample solution: 500 mg of Lonicera japonica Flower Powder in 10 ml methanol, sonicate for 10 min and filter. Evaporate the filtrate at about 50 under reduced pressure to dryness. Dissolve the reidue with 5 ml of water, and then add 10 ml of ethyl acetate, mix. After the solution separated into two layers, take the ethyl acetae layer. Repeat the extraction one more time, combine the ethyl acetate extracts and evaporate the solvent at about 50º under reduced pressure to dryness, dissolve the residue with 2 ml methanol. (See Chromatography <621>, Thin-Layer Chromatography) Adsorbent: Use a chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates) Application volume: 5 µl as 8-mm bands Relative Humidity: Condition the plate to a relative humidity of about 50% using a suitable device Temperature: 25º Developing solvent system: The upper layer solution of the mixture of ethyl acetate, formic acid and water (7:5:5) Developing distance: 6 cm Derivatization reagent: A solution of 3% aluminum chloride in ethanol
2 Samples: Standard solution A, Standard solution B, and Sample solution Apply the samples as bands to a suitable high performance thin-layer chromatographic plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry in air. Derivatize with Derivatization reagent, heat at 105º for 5 min, and examine immediately under UV light at 366 nm. System suitability: Under UV light at 366 nm, the chromatogram of Standard solution B exhibits four bands in the lower-third section; one blue fluorescent band corresponds in Rf to the band due to chlorogenic acid in the chromatogram of Standard solution A; one yellow band above chlorogenic acid corresponds in Rf to the band due to luteolin-7-o-glucoside in the chromatogram of Standard solution A; another faint blue fluorescent band is below chlorogenic acid; another yellow band below the faint blue fluorescent band is due to rutin. Standard solution B exhibits three blue fluorescent bands in the middle-third section of the chromatogram; the blue fluorescent band with a highest Rf is due to 3,5-di-O-caffeoylquinic acid; the faint blue fluorescent band with a midle Rf is due to 4,5-di-O-caffeoylquinic acid; another faint blue fluorescent band is below 4,5-di-O-caffeoylquinic acid. Acceptance criteria: Under UV light at 366 nm, the Sample solution chromatogram exhibits an intense blue fluorescent band and a yellow band at Rfs corresponding to the bands due to chlorogenic acid and luteolin-7-o-glucoside in the chromatogram of Standard solution A, respectively. The Sample solution exhibits additional bands corresponding to similar bands in the chromatogram of Standard solution B, these include another one yellow band in lower-third section and two fluorescent bands in middle-third section. C. HPLC : Proceed as directed in the test for Content of Caffeolyquinic Acids. Acceptance criteria: The chromatogram of the Sample solution exhibits an intense peak with a retention time corresponding to chlorogenic acid in Standard solution A, the peaks related to caffeoylquinic acids for neochlorogenic acid, cryptochlorogenic acid, 3,4-di- O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid at retention times corresponding to the same caffeoylquinic acids in the Standard Solution B and the Reference Chromatogram provided with the lot of USP Lonicera japonica Flower Dry Extract RS being used. The content ratios for 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid versus chlorogenic acid are listed in Table 2; the peaks for neochlorogenic acid, cryptochlorogenic acid and 3,4-di-O-caffeoylquinic acid are smaller minor peaks. D. HPLC : Proceed as directed in the test for Content of Iridoids. Acceptance criteria: The chromatogram of Sample solution exhibits a peak with a retention time corresponding to secoxyloganin in Standard solution A, the peaks related to iridoids for sweroside and centauroside at retention times corresponding to the same iridoids in the Standard solution B and the Reference Chromatogram provided with the lot of USP Lonicera japonica Flower Dry Extract RS being used. ASSAY CONTENT OF CAFFEOYLQUINIC ACIDS Solution A: 0.1% Phosphoric acid in water Solution B: Acetonitrile Mobile phase: See Table 1. Table 1 Time (min) A (%) B (%) [NOTE: Protect from light and proceed under low actinic light. The Standard solution and Sample solution are stable for 24 h at room
3 temperature.] Standard solution A: 0.30 mg/ml of USP Chlorogenic Acid RS in methanol Standard solution B: 5.0 mg/ml of USP Lonicera japonica Flower Dry Extract RS in 75% methanol, sonicate for 15 min, and pass through a membrane filter of 0.22 μm or finer pore size. Sample solution: Accurately transfer about 50 mg of Lonicera japonia Flower powder into a suitable stoppered conical flask, accurately add 5 ml of 75% methanol. Weigh the filled flask with a precision of ± 0.1 mg and then sonicate for 30 min. After cooling to room temperature, adjust the weight to initial by adding methanol. Pass through a membrane filter of 0.22 μm or finer pore size, and discard the first portion of the filtrate. (See Chromatography <621>, System Suitability) Mode: LC Detector: UV 327 nm Column: 4.6 mm 25 cm; 5-μm packing L1 (similar to Syncronis C18) Column temperature: 15 ± 1º Flow rate: 0.7 ml/min Injection volume: 2 µl System suitability Sample: 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 Lonicera japonica Flower Dry Extact RS being used. Resolution: NLT 1.5 between the peak of chlorogenic acid and the peak of cryptochlorogenic acid, Standard solution B Tailing factor: NMT 2.0 for the peak of chlorogenic acid, Standard solution A Relative standard deviation: NMT 2.0% for chlorogenic acid, Standard solution A 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 Lonicera japonica Flower Dry Extract RS being used, identify the retention times of the peaks corresponding to neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid. The approximate relative retention times (RRT), relative to chlorogenic acid, are provided in Table 2. (The RRTs are variable due to different column, mobile phase flow rate and so on.) Table 2 Analyte Approximate Relative Retention Time Conversion Factor Content Ratio neochlorogenic acid minor peak* chlorogenic acid cryptochlorogenic acid minor peak* 3,4-di-O-caffeoylquinic acid minor peak* 3,5-di-O-caffeoylquinic acid ,5-di-O-caffeoylquinic acid *The peak area is lower than the peak area for 4,5-di-O-caffeoylquinic acid Separately calculate the percentage of chlorogenic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid in the portion of Lonicera japonica Flower taken: Result = (r U /r S ) C S (V/W) F 100 r U = peak area of relevant analyte from the Sample solution r S = peak area of chlorogenic acid from the Standard solution A C S = concentration of USP Chlorogenic Acid RS in the Standard solution A (mg/ml) V = volume of the Sample solution (ml) W = weight of Lonicera japonica Flower taken to prepare the Sample solution (mg) F = conversion factor for the analytes as provided in Table 2.
4 Calculate the content of caffeoylquinic acids as the sum of chlorogenic acid, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid. Acceptance criteria: NLT 3.8% of caffeoylquinic acids calculated as the sum of chlorogenic acid, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid on the dried basis CONTENT OF IRIDOIDS Solution A, Solution B, Mobile phase, Standard solution B and Sample solution are prepared in the same way as in the test for Content of Caffeoylquinic Acids. Standard solution A: 0.05 mg/ml of USP Secoxyloganin RS in methanol (See Chromatography <621>, System Suitability) Mode: LC Detector: UV 240 nm Column: 4.6 mm 25 cm; 5-μm packing L1 (similar to Syncronis C18) Column temperature: 15 ± 1º Flow rate: 0.7 ml/min Injection volume: 2 µl System suitability Sample: 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 Lonicera japonica Flower Dry Extact RS being used. Resolution: NLT 1.5 betwen the peak of secoxyloganin and the peak before it, Standard solution B Tailing factor: NMT 2.0 for the peak of secoxyloganin, Standard solution A Relative standard deviation: NMT 2.0% for secoxyloganin, Standard solution A 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 Lonicera japonica Flower Dry Extract RS being used, identify the peaks of sweroside, secoxyloganin, and centauroside. The approximate relative retention times for the peaks of sweroside, secoxyloganin, and centauroside are 0.85, 1.00 and 1.80, respectively. Calculate the percentage of secoxyloganin, sweroside and centauroside in the portion of Lonicera japonica Flower Powder taken: Result = (r U /r S ) C S (V/W) F 1100 r U = peak area of relevant analyte from the Sample solution r S = peak area of secoxyloganin from the Standard solution A C S = concentration of USP Secoxyloganin RS in the Standard solution A (mg/ml) V = volume of the Sample solution (ml) W = weight of Lonicera japonica Flower Powder taken to prepare the Sample solution (mg) F = conversion factor for analytes (1.00 for secoxyloganin, 1.03 for sweroside, and 0.89 for centauroside) Calculate the content of iridoids as the sum of secoxyloganin, sweroside and centauroside. Acceptance criteria: NLT 0.80% of iridoids calculated as the sum of secoxyloganin, sweroside and centauroside on the dried 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 Copper: NMT 20.0 µg/g ARTICLES OF BOTANICAL ORIGIN, General Method for Pesticide Residues <561>: Meets the requirements.
5 MICROBIAL ENUMERATION TESTS <2021>: The total aerobic bacterial count does not exceed 10 5 cfu/g, the total combined molds and yeasts count does not exceed 10 3 cfu/g, and the bile-tolerant Gram-negative bacteria does not exceed 10 3 cfu/g. TESTS FOR SPECIFIED MICROORGANISMS <2022>: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli. ARTICLES OF BOTANICAL ORIGIN, Aflatoxins <561>: Meets the requirements. SPECIFIC TESTS Limit of Triterpenoid Saponins by TLC Standard solution: 1.0 mg/ml of USP macranthoidin B RS, and 1.0 mg/ml USP dipsacoside B RS in methanol. Sample solution: 500 mg of Lonicera japonica Flower powder in 10 ml methanol, sonicate for 10 min and filter. (See Chromatography <621>, Thin-Layer Chromatography) Adsorbent: Use a chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates) Application volume: 3 µl as 8-mm bands Relative Humidity: Condition the plate to a relative humidity of about 50% using a suitable device Temperature: 25º Developing solvent system: The upper layer solution of a mixture of n-butanol, formic acid and water (4:1:5) Developing distance: 6 cm Derivatization reagent: A solution of 10% sulphuric acid in ethanol Samples: Standard solution and Sample solution Apply the samples as bands to a suitable high performance thin-layer chromatographic plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry in air. Derivatize with Derivatization reagent, heat at 105º for 5 min, and examine immediately under UV light at 366 nm. System suitability: Under UV light at 366 nm, the chromatogram of Standard solution exhibits two clearly separated bands in the lower-third section, the band with a higher Rf is due to dipsacoside, the band with a lower Rf is due to macranthoidin B. Acceptance criteria: Under UV light at 366 nm, the Sample solution chromatogram do not exhibits bands at Rfs and colour corresponing to the bands due to dipsacoside and macranthoidin B in the chromatogram of Standard solution; the Sample solution chromatogram do not exhibits bands with similar color as the bands of dipsacoside and macranthoidin B and with Rfs between the bands of dipsacoside and macranthoidin B. ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives, Method 1 <561>: NLT 30.0% ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives, Method 2 <561>: NLT 35.0% LOSS ON DRYING <731>: : Dry 2 g of Lonicera japonica Flower Pwder, at 105º for 2 h. Acceptance criteria: NMT 12.0% ARTICLES OF BOTANICAL ORIGIN, Total Ash <561> : 4 g of Lonicera japonica Flower Powder Acceptance criteria: NMT 10.0% ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash <561>: : 4 g of Lonicera japonica Flower Powder Acceptance criteria: NMT 3.0% ADDITIONAL REQUIREMENTS PACKAGING AND STORAGE: Preserve in well-closed containers, store at a cool and dry place, protect from light, moisture and moth. LABELING: The label states the Latin binomial and the part(s) of the plant contained in the article. USP REFERENCE STANDARDS <11> USP Lonicera japonica Flower Dry Extract RS USP Secoxyloganin RS USP Chlorogenic Acid RS USP Luteolin-7-O-Glucoside RS USP Dipsacoside RS USP Macranthoidin B RS
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Lonicera japonica Flower Dry Extract. Proposed For Development Version 0.1. Published on Herbal Medicines Compendium (
Published on Herbal Medicines Compendium (https://hmc.usp.org) Lonicera japonica Flower Dry Extract Proposed For Development Version 0.1 Lonicera japonica Flower Dry Extract DEFINITION The article is prepared
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