Homeopatically produced preparation from Juglans nigra L. shell: the analysis of biologically active compounds and the antioxidant activity study

Preparations referred to as homoeopathic are those made of homoeopathic raw materials using homoeopathic manufacturing procedures. In order to measure biological activity of homoeopathic preparations scientists use the same investigation methods as with other kinds of chemical products [30]. For this study homoeopathic tincture of black walnut (Juglans nigra L.) shell and its D1and C1 dilutions have been selected as these produce low-grade solutions in which active substances still can be identified and concentrations as well as bioactivity can be assessed by available methods of testing. The scientific literature sources state that the black walnut shell contains nine basic chemical substances [29]. It is found that one of the basic active ingredients is juglone which possesses cytotoxic properties effective against melanoma, the liver, lung, heart and blood cells. [25,28,38,39]. As the studies of black walnut preparations are not so common, we have chosen to investigate the juglone amount and polyphenolic compounds in solutions and to measure their antioxidant properties. The aim of the research: To produce homoeopathically original tincture of the black walnut shell and its D1 and C1 dilutions, to explore polyphenolic compounds, the level of juglone and evaluate their antioxidant activity. The methodology: Original homoeopathic tincture (urtincture) and its solutions were produced following the specification of Homoeopathy Pharmacopoeia (HAB 2000, rule 3a). Spectrophotometrically was tested the amount of polyphenolic compounds using the Folin – Ciocalteu reagent. From the calibration curve of gallic acid the amount of polyphenolic compounds, expressed as mg/ml GRE was measured. Spectrophotometrically was measured the juglone concentration in the investigated solutions. From the calibration curve of juglone the amount of this substance was measured in the tested solutions [37]. In order to determine the antioxidant activity a fluorimetric method was applied. For the statistical data analysis we used the program SigmaPlot version 9.0 (Systat Software Inc.). The results of the study: The raw substance (Juglans nigra L. nutshell), picked at Kaunas Botanical Garden in 2012, was used to produce original homoeopathic tincture and its D1, C1 dilutions. In the original tincture the measured amount of phenolic compounds was 13,36±0,1mg/ml GRE, in homoeopathically prepared D1 solutions it was 1,68±0,05mg/ml GRE, and in C1 solutions -0,46±0,03mg/ml GRE. After spectrophotometric study of juglone amount, it was found that in the homoeopathic tincture of black walnut the juglone content comprised 2,0±0,2µg/ml, in homoeopathic D1 solutions - 0,7±0,1µg/ml, and in C1 solutions - 0,2±0,1µg/ml of juglone. The antioxidant activity studies have shown the amount H2O2 being neutralized by the solutions used for testing. For the concentration of original tincture (0,64mg/ml GRE) 30 percent reduction of H2O2 amount was found, further 50 percent (1, 27mg/ml±5,8) of the total amount, 70 percent (1,91mg/ml±4,5) of the total amount. D1 dilution neutralizes 40 percent (0,52mg/ml±2,5) of the total amount, 50 percent (1,04 mg/ml±2,5) of the total amount, 60 percent (1,56mg/ml±9,5) of the total amount. C1 solution was not marked by antioxidant activity. Conclusions: 1. The produced homoeopathic tincture of the black wall nut and its D1 and C1 dilutions together with the measured and compared amount of polyphenol compounds and juglone amount in the tested solutions indicate that in the homoeopathic preparations of such dilution level, the active substances and their concentration can still be measured by applying the standard methods of investigation. 2. After evaluation and comparison of antioxidant activity of the tested solutions it was found that homoeopathic tincture was marked by the highest activity neutralizing H2O2, and homoeopathic C1 dilution did not show the neutralizing effect of H2O2. The research outcomes indicate that the studied homoeopathic preparations are characterised by the active compounds concentration- dependent antioxidant activity, while C1 dilution does not possess enough active ingredients to exhibit this activity