Биологические науки / 2.Структурная ботаника и биохимия растений

Galkin O.Yu., Kotov A.G.

National Technical University of Ukraine “Kyiv Polytechnic Institute”, Ukraine;

Ukrainian Research Pharmacopeial Centre for Medicines Quality, Ukraine

Pharmacognosical Study and STANDARDIZATION of hop cones (HUMULUS LUPULUS L.)

The aim of our work was to conduct pharmacognosical study and standardization of hop cones (Humulus Lupulus L.), as well as recommendations for the formation of analytical documentation (AD) for raw material.

AD is given respectively of European Pharmacopoeia (EuPh) (2005), Monograph “Hop strobile”.

Based on the analysis of plant materials collected in the years 2006-2007, we offer to put in the wording of EuPh following chapters of AD: “External characters” and “Identity. Microscopy”.

The chapter “Identification. Xanthohumol, humulones, lupulones” we offer to put in the wording of EuPh method.

Impurities content – no more than 2% (other parts of the plant, organic or mineral). Determination is hold by gravimetric method: an analytical sample of raw material (100 g) placed on a smooth surface and a clean spatula or tweezers separating impurities. Each kind of impurity weighed separately and calculates the content.

Assay. Substances extracted with alcohol (70% v/v). All analyzed a series of raw materials meet the requirements regulated EuPh – not less than 25%.

Below methods of quality control of herbal drug.

Appearance. Hop cones are predominantly solitary, 2 cm to 5 cm long, petiolate, egg-shaped, comprising multiple oval greenish-yellow, sessible, sheet, flattened and symmetrical leaves forming tile-covering. Floral disk is asymmetrical because of a folding around a seed bearer covered with petals. Seed-bud or, sometimes, fruit, base of covering leaves, floral disk and, in particular, folds are covered with small orange-yellow glandules.

Identification (microscopy). Powder the raw material (355). Examine the obtained greenish-yellow powder microscopically by using a solution of chloral hydrate R. The following constituents can be seen in the powder: fragments of flower disks and covering leaves formed of polygonal random-shaped epidermal cells with curled membranes; unicellular conical, linear or bent covering hairs with thin smooth membranes; rarely – stomatal apparatus of anomocytic type; mesophyll fragments with small druses of calcium oxalate; many characteristic orange-yellow glandular hairs with short bicellular legs carrying concave leaf expansion of 150 mm to 250 mm in diameter comprising hemispherical layer of secretory cells with cuticle separated and swelled with the accumulated essential oil and resin; and fragments of elongated cells of the sclerenchyma of seed-cover with thick membranes containing grooves and multiple pores.

Identification (xanthohumol, humulones, lupulones). Testing is performed by using thin layer chromatography (TLC) (in accordance with the National Pharmacopoeia of Ukraine (NPhU),1^st edition, 2.2.27).

Test solution. Transfer 1.0 g of grinded raw material obtained under “Extractives” Section into a 20 mL flask, add 10 mL of water R - methanol R mixture (3:7) and agitate for 15 min. Filter the content of the flask into a 20 mL flask.

Reference solution. Transfer 1 mg of sudan orange R, 2 mg of curcumine R and 2 mg of dimethylaminobenzaldehyde R in a 10 mL flask, dissolve in 10 mL of water R - methanol R mixture (3:7) and mix.

Apply 10 × 3 mm bands of 20 mL of each of the test solution and reference solution on a start line of 6x20 cm Kieselgel 60 F₂₅₄ TLC plate (Merck). Dry the plate in the air for 10 min, put the plate into a chromatographic chamber with a mixture of solvents: cyclohexane R – ethyl acetate R – glacial acetic acid R (60:38:2) and run chromatograms by using the ascending method. Remove the plate from the chromatographic chamber when the solvent front advance approximately by 15 cm and dry the plate in the air under exhaust hood for 15 min.

Examine the plate in UV light (254 nm): the chromatogram of the reference solution contains three dark spots: low intensity spot of curcumine in the lower quarter, dimethylaminobenzaldehyde spot somewhat below the chromatogram centre and sudan spot – above the chromatogram centre. The chromatogram of the test solution should contain: slightly dark spot almost on the level of curcumine spot on the chromatogram of reference solution (xanthohumol), humulone spots – almost on the level of dimethylaminobenzaldehyde spot and lupulone spots – almost on the level of sudan spot.

Examination of the chromatographic plate of test solution in UV light at 365 nm should reveal blue fluorescence of lupulone spots, brown fluorescence of humulone spots and dark-brown fluorescence of xanthohumol spot.

Spray the chromatogram with Folin-Ciocalteu reagent R (a mixture of phosphomolybdate and phosphotungstate) and place it in a chamber saturated with ammonia vapour, and then examine in the day light. Humulone and lupulone spots in the chromatogram of test solution turn blue-grey and xanthohumole spot – green-grey.

Appearance of other spots of different size and colour is acceptable.

Weight loss on drying. Not more than 12 % (NPhU,1^st edition, 2.2.32).

Total ash. Not more than 12 % (NPhU,1^st edition, 2.4.16).

Impurities. (NPhU,1^st edition, 2.8.2) Not more than 2 %.

Extractives. Powder the raw material (355). Transfer approximately 10.00 g of accurately weighed raw material in a 1000 mL flask, add 300.0 mL of 70 % alcohol R, connect a reflux condenser and heat in a water bath for 10 min. Cool down the mixture and filter it discarding first 10 mL of the filtrate. Evaporate 30.0 mL of the obtained filtrate and dry the residue at 100 °С to 105 °С for 2 h. Using the formula:

X = [300 × (m₁ – m₀) × 100 × 100] / [m × (100 – W) × 30],

here: m – weight of a sample of raw material, g; m₁ – weight of a weighing bottle with residue, g; m₀ – weight of an empty weighing bottle, g.

Percentage of extractives should be not less than 25 %.

Conclusion: the obtained data is the basis for the formation of analytical documentation for raw materials.