Development of Purified Propolis Extract Technology, Analysis and Evaluation of Antimicrobial Activity
Dangoule Vansevičiūtė, JSC “
Valentis” Vilnius, Lithuania
E-mail:
povilonyte@valentis.ltPresented at: 5th German Apitherapy Congress, March 23-25, 2007, Passau, Germany
Propolis (bee glue) and its preparations are widely used in medical practice, due to their antimicrobial, anesthetic, immunity boosting effect, they act against fungus, suppress inflammation , promote synthesis of antibodies , facilitate healing of wounds and ulcers.
It was identified that propolis ethanol extract of as little as 0.08 percent concentrations prevents the growth of microorganisms. Preparations containing propolis not only have a wide antimicrobial effect, but, different than with intake of antibiotics, resistant microorganism forms do not develop while using the preparations containing propolis.
From the chemical point of view, propolis is a very complex compound. Propolis contains over 200 different chemical components. These are polyphenoles (chalcones, flavones, flavonones, flavonoles), aromatic acids, terpenoids, alcohols, aldehydes, ketones, amino acids, sugars, vitamins, minerals, waxes, fatty acids, steroids etc. Antimicrobial effect of propolis extract is determined by flavonoids, caffeic, cinnamic, benzoic acids contained in the extract, and other chemical compounds, their amount and their synergistic interaction .
JSC “Valentis” began propolis research in 2005. The goal of work was to produce the purified soft propolis extract, to perform its chemical analysis, to research the antimicrobial effect and to use it as an antimicrobial conservation agent in medication manufacture.
We developed the manufacturing technology of purified propolis extract. Manufacture consisted of several stages. During the course of manufacture, the concentration, temperature, pressure of extractant (ethanol) was being changed.
We manufactured 5 batches of purified propolis extract. The purified propolis extract is a soft material of dark brown colour of specific odour, it easily and without residue dissolves in 96 percent V/V ethanol. Dry residue of extract is 80 ± 5 %. We did not find waxes in the purified propolis extract. Its oxidation rate did not exceed 10 sec.
Unpurified propolis extract contained 3 ± 0.6 % of waxes, oxidation rate did not exceed 22 sec. We identified the polyphenolic compounds (precipitate of yellow colour) qualitatively with the solution of lead acetate, flavonoids (precipitate of green colour) – with the solution of aluminum trichloride, flavonone pinostrobin was identified by a method of thin-layer chromatography (solvent system: chloroform – 96 percent V/V ethanol 90:10). We developed the spots in UV light at a 254 nm wavelength, and afterwards with diazotized sulfanilic acid.
Quantitatively we identified the phenolic compounds by a spectrophotometric method, by measuring the absorption amount of ethanol solution in the wavelength of 290 nm. We found that the extract should contain 45 ± 3 % (n = 5) of phenolic compounds. By making the coloured compound with Folin – Ciocalteu agent and by measuring the absorption rate in the wavelength of 760 nm, we identified the amount of polyphenolic compounds, recalculating them into gallic acid. We found that the extract must contain 0.7 ± 0.05 % (n = 5) of polyphenolic compounds recalculated into gallic acid. The specific component in propolis is flavonone pinostrobin. Its amount was identified by a spectrophotometric method, by measuring the absorption amount of ethanol solution in the wavelength of 289 nm. We found that the extract must contain 25 ± 1 % (n = 5) pinostrobin.
The amounts of active substances in the unpurified propolis extract differed very insignificantly. We analyzed the antimicrobial activity of purified propolis extract. We used 9 reference cultures of microorganisms: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 33499), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 12459), Bacillus subtilis (ATCC 6623), Bacillus cereus (ATCC 8035) and Candida albicans (ATCC 60193).
We estimated the antimicrobial activity by the dilution method, using the Muller – Hinton broth and 0,9 % solution of sodium chloride. We made 5 dilutions up to 3200 times. We found that after diluting the extract by 400 times (concentration of purified propolis extract is 0.044 mg %), it completely suppresses the growth and proliferation of the above cultures. After diluting the extract by 800 times (concentration of purified propolis extract is 0.011 mg %), it suppresses the growth and proliferation of the above cultures, except Escherichia coli and Proteus mirabilis. After diluting the extract by 1600 times and more, its antimicrobial activity was not manifested anymore.
Due to its strong and wide antimicrobial effect, the propolis extract can be used as antimicrobial conservation agent in manufacture of different medications, replacing the substances of chemical nature.
Currently JSC “Valentis” is in the stage of research of the stability of different syrups, where the purified propolis extract is used as antimicrobial conservation agent. The research is in progress.
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