From functional food to medicinal product: systematic approach in analysis of polyphenolics from propolis and wine

Abstract

In the last decade we have been working on standardization of propolis extract and determination of active constituents of wine those are rich in polyphenolics and have nutritional as well as therapeutic value. Here we are summarizing our results and providing overview on systematic approach how to analyse natural products rich in flavonoids and phenolic acids.Chromatographic methods (thin layer chromatography and high performance liquid chromatography) were used for identification, quantification, and characterization of individual flavonoid or phenolic acid. Total content of active constituents and antioxidant activity were determined by spectrophotometry. Pharmacokinetic parameters were determined by high performance liquid chromatography and using appropriate software. Quantitative structure-activity relationship study of antioxidant activity was conducted, as well as assessment of prolonged propolis supplementation on antioxidative status of organism.Thin layer chromatography-densitometry has been proven as quick and reliable method for standard analysis of propolis and wine; the best mobile phase being chloroform - methanol - formic acid (98-100%) in ratio 44 : 3.5 : 2.5 (v/v). Higher number of polyphenolics was determined by high performance liquid chromatography; 15 compared to 9 by thin layer chromatography. Interactions in situ with acetylsalicylic acid were detected with most of polyphenolics analysed. Plasma protein binding and blood-barrier penetration was greatest for flavone. The interactions with human serum albumin have been grater than 95% for all flavonoids analysed. The prolonged propolis consumption increased superoxide dismutase activity.The necessity of standardization of natural products and their registration as functional nutraceuticals demand easy, quick and inexpensive methods of analysis. In this work we provided overview of analytical part for polyphenolics that could be used as data for possible registration of final products either as functional food or medicinal product.This feature introduces the readers to the authors' research through a concise overview of the selected topic. Reference to important work from others in the field is included.

Figure 1

Basic structures of flavonoids (A) and phenolic acids (B-cinnamic acid, C-benzoic acid).

Figure 2

The map of Croatia. Areas of origin of propolis samples (marked by numbers).

Figure 3

The structure of stationary phase of immobilized artificial membrane – phosphatidylcholine – drug-discovery column (IAM.PC.DD).

Figure 4

Dendrogram for eleven TLC mobile phases used fot determination of polyphenols in samples of Croatian propolis.

Figure 5

2D-TLC of caffeic acid in propolis sample from Peruča and standards of caffeic acid. The plate was first developed in the direction 1 then 2 using mobile phase 1 (n-hexane : ethyl acetate : glacial acetic acid = 31 : 14 : 5 v/v) and mobile phase 2 (chloroform : methanol : formic acid = 44 : 3.5 : 2.5 v/v). Bands were visualized under short and long wave UV light and under long wave UV light after spraying with 1% AlCl₃ ethanol solution. Concetration obtained from chromatograms: a = m_max = 2.50 μg, b = m_min = 0.83 μg, c = m_mid = 1.67 μg, PS = m_{propolis sample} = 2.91 μg [18].

Figure 6

Structures of flavonoids and phenolic acids quantified in propolis tinctures.

Figure 7

Chromatogram of interaction of flavonoid pinocembrin and ketoprofen. Interaction of flavonoid pinocembrin and ketoprofen obtained under long wave UV light. SF – standard of flavonoid (0.33 mg/mL), A, B, C – interaction lines of pinocembrin and ketoprofen in concentration 0.5, 1.0 and 1.5 mg/mL respectively, SL – standard of ketoprofen in the same concentration as in A, B and C.

Figure 8

Band-blot test of polyphenols present in propolis using the stable free radical DPPH·. Chromatogram obtained after spraying the layer (applied polyphenol standard mixture bands in the range from 45 (track 1) to 10 μg/mL (track 8)) with 0.3 mM solution of DPPH^· radical. AOE of standard mixture is 1.89 (H = 1.89c + 22.856, r² = 0.974) [22].

Figure 9

Chromatograms of extracts of red wines "Merlot" (1) and "Frankovka" (2) by use of mobile phase benzene : ethyl acetate : formic acid = 30 + 15 + 5 v/v/v. a) recorded under UV light λ = 254 nm; b) recorded under UV light λ = 366 nm, after spraying with 1% ethanol solution of AlCl₃. The standard substances used: A – gallic acid, B – caffeic acid, C – apigenin, D – kaempferol, E – p-coumaric acid, F – naringenin [23].

Figure 10

TLC identification and quantification of gallic acid. Photograph of the chromatographic plate recorded at UV light λ = 254 nm, for the purpose of quantification of gallic acid in the sample of wine, with the spectrum of marked peaks obtained using CAMAG densitometer.

Figure 11

Structure and differences in HSA binding of enantiomers.

Figure 12

HPLC chromatogram of wine sample Postup registered at 280 nm.

Figure 13

Total polyphenols in wine samples as equivalents of gallic acid (GAE, mg/L) [5]. Codes for wine samples: malv – Malvazija; tram – Traminac; pelj – Pelješac; zwei – Zweigelt; merl – Merlot; fran – Frankovka; cabs – Cabernet Sauvignon; borg – Borgonja; pinc – Pinot crni; ding – Dingač; post – Postup; plav – Plavac.

Figure 14

Structures of catechin and epicatechin.

Figure 15

Antioxidant activity of propolis in vivo. Activity of superoxide-dismutase (SOD) (U/g Hb) during the study: a) in the men test group and b) in the women test group. Data are expressed as means ± S.D.; B (1) represents the baseline on the 15^th day prior to the study and PS (30) represents propolis supplementation on the 30^th day. The symbol (*) denotes statistically significant change [29].