Investigations on its antioxidative and anti-inflammatory potential

Transcription

1 - 1 - CITROZINE Investigations on its antioxidative and CITROFRESH SUPERCONCENTRATE anti-inflammatory potential Investigator and responsible for the correctness of the test protocol, results, conclusions and application of GLP guidelines: Schongau/Germany November 12, Prof. Dr. Peter C. Dartsch

2 - 2 - Summary CitroZine, a highly concentrated technological product that is used as an ingredient to Personal and HealthCare industries for a wide range of applications, was examined for its antioxidant (cell-free test assay) and anti-inflammatory (cell-based test assay) potential. The results are: 1 Dose-dependent antioxidative effect of CitroZine Inactivation of free exogenous superoxide anion radicals was approximately 100% for concentrations 250 µg/ml (= vol%). Under these circumstances the unwanted interactions between CitroZine and the used tetrazolium dye which take place at concentrations 1,000 µg/ml (= 0.1 vol%) did not falsify the results. The EC50, i.e. the concentration resulting in a 50% inactivation of free superoxide anion radicals, was calculated to be 175 µg/ml which is equivalent to a concentration of approximately 0.02 vol%. As seen in Table S1, CitroZine was the best natural product/mixture tested in this respect. Table S1: Comparative antioxidative EC50 values for various test substances or extracts as obtained under similar test conditions as described here. Ascorbic acid is synthetically produced (CAS No ) and was used in analytical grade (p.a.) purity. Test substance or extract EC50 (antioxidative ) in µg/ml Ascorbic acid p.a. 25 CitroZine TM 175 Red wine (Dornfelder) 275 Microalgae & mineral formulation 500 Aronia extract (dry powder) 600 Dandelion extract (fresh) 2,000 Cranberry extract (dry powder) 2,000 Cystus extract (dry powder) 2,750 Rosemary extract (fresh) 3,000 Basil extract (fresh) 3,500 2 Dose-dependent anti-inflammatory potential of CitroZine (energy metabolism) Evaluation of the anti-inflammatory potential of CitroZine by reducing the energy metabolism of unstimulated functional neutrophils, i.e. inflammation-mediating cells, showed that concentrations up to 100 µg/ml (= 0.01 vol%) caused a specific metabolic reduction suggesting a reduced migration and oxygen radical production in the inflamed area. At concentrations 250 µg/ml, the acidic ph of CitroZine caused a marked loss in cell vitality. The EC50, i.e. the concentration resulting in a 50% inhibition of energy metabolism of unstimu-

3 - 3 - lated functional neutrophils, was calculated to be 150 µg/ml which is equivalent to a concentration of about vol%. 3 Dose-dependent anti-inflammatory potential CitroZine (oxidative burst) Inactivation of superoxide anion radicals generated by PMA-stimulated functional neutrophils in the course of an oxidative or respiratory burst showed a strong anti-inflammatory potential of CitroZine at concentrations which did not affect cell vitality due to acidic ph. This strong anti-inflammatory potential might be related to the bioflavonoid content of Citro- Zine. The EC50, i.e. the concentration resulting in a 50% inactivation of superoxide anion radicals generated by stimulated functional neutrophils, was calculated to be 130 µg/ml which is equivalent to a concentration of about vol%. As seen in Table S2, CitroZine was the best natural product/mixture tested in this respect. Table S2: Comparative anti-inflammatory values for various test substances or extracts as obtained under similar test conditions as described here. Ibuprofen sodium salt is synthetically produced (CAS No ) and was used in analytical grade (p.a.) purity. Test substance or extract EC50 (anti-inflammatory ) in µg/ml Ibuprofen sodium salt p.a. 50 CitroZine TM Red wine (Dornfelder) Bilberry extract (dry powder) 1,000 Dandelion extract (fresh leaves) 2,500 Cranberry extract (dry powder) 2,500 Aronia extract (dry powder) 5,000 Coltsfoot extract (fresh leaves) 10,000 In conclusion, CitroZine possesses a strong antioxidant and anti-inflammatory potential by inactivating superoxide anion radicals and reducing the energy metabolism of inflammation-mediating cells at concentrations which are markedly below the manufacturer s recommendation of 0.3 to 0.5 vol%. From the results, CitroZine can be recommended as an excellent tool against oxidative stress and oxygen radical-mediated inflammatory processes.

4 - 4 - Background Our body constantly reacts with oxygen as part of the energy producing processes of cells. As a consequence, the vast majority of reactive oxygen species (ROS) come from endogenous sources as by-products of normal and essential metabolic reactions. To counteract the effects of ROS in vivo, there are a number of distinct antioxidant mechanisms within the cells which neutralise ROS under normal conditions. Oxidative stress and the associated damage to cellular lipids, proteins and DNA results when these compensatory mechanisms fail to detoxify increasing loads of ROS. Elevated levels of ROS, which contribute to a decline in cellular function, have been reported to coincide with a number of human pathologies including chronic inflammatory diseases, cancer, cardiovascular diseases, ocular degeneration and neurological disorders. The oxidative damage has also been implicated to have an impact on the body's aging process (Figure 1). Therefore, a valid and sensitive screening as represented by TIIOS is a useful tool to examine the antioxidant and/or anti-inflammatory potential of test substances. For the basic principle of this test, see also Figure 2. Figure 1: Human pathologies related to reactive oxygen radicals (ROS) and oxidative stress.

5 - 5 - Product description and manufacturer CitroZine is a highly concentrated unique technological product that is used as an ingredient to Personal and HealthCare industries for a wide range of applications. CitroZine is composed of demineralised water, citric acid, malic acid, glycerine and citrus amara aurantium extract. All ingredients are natural and food grade. It is manufactured by GDM Technologies Pty Ltd, 2/55 West Fyans Street, Newtown Victoria, Australia Aim of the study and test concentrations Aim of the study was to investigate whether CitroZine is able to (1) inactivate free exogenous oxygen radicals (superoxide anion radicals; antioxidant effect) (2) reduce energy metabolism and inactivate superoxide anion radicals generated by inflammation-mediating cells (functional neutrophils; anti-inflammatory effect) A concentration of 0.3 to 0.5 vol% of CitroZine is recommended by the manufacturer which is equivalent to a concentration of 3 to 5 mg/ml. In order to cover a wide range in the cell-free test assays, the test concentrations varied from 0.01 to 10 mg/ml; in the cell-based test assays the concentrations were adapted due to cell vitality depending on the effective neutralisation of the acidic ph by using HEPES-buffer system. Table 1: Stock solutions and corresponding test concentrations in vol% and µg/ml for CitroZine Stock solution x10 (concentration in vol%) Stock solution x10 (concentration in µg/ml) ,000 25,000 10,000 5,000 2,500 1, Test concentration in vol% Test concentration in µg/ml 5,000 2,500 1, Antioxidant screening in the cell-free test assay (scavenger effect) In contrast to the cell-based test assay described below, this cell-free test assay uses potassium superoxide (1 mg/ml) as a donor for superoxide anion radicals. The efficacy of radical inactivation by CitroZine was monitored by the radical-induced cleavage of WST-1, a red tetrazolium dye which yields a highly water-soluble yellow formazan dye upon cleavage. According to the dye used, optical density at nm (delta OD) was monitored using an ELISA reader. In order to exclude any falsification by unwanted interactions between WST-1 and CitroZine, it was also examined simultaneously that the test substance did not cause any alterations in optical density during the observation periods without addition of superoxide anion radicals.

6 - 6 - Basic process temperature: ELISA reader: Reading parameters: Data presentation & analysis: 37 ± 0.1 C BioTek Elx808 with software module KC-4 For 60 minutes with one reading every minute after shaking Correction of the optical density measured at nm (delta OD) by the corresponding blank values and presentation of curves. For data analysis, the linear curve increase during the first 10 minutes after addition of superoxide anion radicals was used (mean V in mod/min). Anti-inflammatory screening in the cell-based test assay (TIIOS) TIIOS is a cell-based test assay which uses the formation of intracellular superoxide radicals of activated human inflammation-mediating cells (functional neutrophils) as an inflammation model to investigate the efficacy of biologically active substances for the inactivation of oxygen radicals. Upon stimulation with phorbol-12-myristate-13-acetate (PMA) functional neutrophils produce superoxide radicals by an oxidative or respiratory burst. The efficacy of radical inactivation by Citro-Zine was monitored by the cleavage of WST-1. In order to exclude false positive results due to cell death and not to radical-inactivating effect, an evaluation of cell vitality was also done. For that purpose, the mitochondrial dehydrogenases activity of differentiated HL60 cells exposed for 60 min to the various concentrations of CitroZine was examined. Moreover, CitroZine might influence the metabolism of inflammation-mediating cells resulting in (1) a reduced migration of these cells into the inflamed area and (2) a reduced formation of superoxide anion radicals. In order to test this aspect of anti-inflammatory action, the energy metabolism of functional neutrophils without and with various concentrations of CitroZine was also monitored by the cleavage of WST-1. All cell-based test assays were undertaken in media containing 20 mm HEPES buffer to neutralise the acidic ph of CitroZine. Annotation: For detailed information on the test principle of TIIOS, please see Dartsch, P.C.: TIIOS a sensitive and cell-based test assay for the screening of biologically active substances for their antioxidant potential. Innovations in Food Technology, 32: (August 2006). Cell line used: Human promyelocytes, cell line HL60, ECACC Three different pools from three different cell batches with different in vitro-age (passage 18-24) were used for the test Differentiation process: Dimethylsulfoxide-induced differentiation to inflammationmediating cells (functional neutrophils) Basal culture medium: RPMI 1640 with L-glutamine containing 5% fetal bovine serum, 100 U/ml of penicillin and 100 µg/ml of streptomycin

7 - 7 - Human monocytic cell (differentiated and activated to functional neutrophil) PMA activation Formation of superoxide anion radical by oxidative burst Anti-inflammatory substances Cleavage of a tetrazolium dye (WST-1) to water-soluble formazan Colorimetric measurement of optical density (delta OD nm) Figure 2: Schematic presentation of the basic principle of TIIOS for the screening of antioxidant and/or anti-inflammatory potential of test substances. Cell density: Primary control of ox. burst: Process temperature: ELISA reader: Reading parameters: Data analysis: cells/ml in phosphate-buffered saline with calcium and magnesium (PBS+), glucose & 20 mm HEPES buffer. In order to avoid ph effects due to the acidity of CitroZine, HEPES buffer was added to the medium Dose-dependent inhibition of oxidative burst by superoxide dismutase (SOD) upon PMA stimulation 37 ± 0.1 C BioTek Elx808 with software module KC-4 For 60 minutes with one reading every minute after shaking Correction of the optical density measured at nm (delta OD) by the corresponding blank values and in relation to untreated controls with (examination of oxidative burst) and without (examination of energy metabolism) PMA stimulation. Depending on the test assay, data were examined for each point of measurement or the linear increase in optical density was used (mean V in mod/min).

8 - 8 - Results and conclusions 1 Antioxidant potential (scavenger effect in cell-free test assay) As depicted in Figure 3 for two single test assays, CitroZine caused a clear dosedependent antioxidant effect by the inactivation of free exogenous superoxide anion radicals. At test concentrations 1,000 µg/ml, the addition of CitroZine to the dye caused a cleavage of the tetrazolium salt and a dose-dependent decrease in optical density (Figure 4). Therefore, this interaction had to be taken into account and a test concentration of maximally 500 µg/ml was concluded to reflect the real situation and to be artifact-free. However, this limitation does not reduce the experimental significance, because even lower concentrations of 250 µg/ml resulted in a nearly complete inactivation of free superoxide anion radicals (Figure 5). The EC50, i.e. the concentration resulting in a 50% inactivation of free exogenous superoxide anion radicals was calculated to be 175 µg/ml. Comparative antioxidative data of reference substances or extracts are given in Table 2 demonstrating that CitroZine was the best natural product/mixture tested in this respect. Table 2: Comparative antioxidative EC50 values for various test substances or extracts as obtained under similar test conditions as described here. Ascorbic acid is synthetically produced (CAS No ) and was used in analytical grade (p.a.) purity. Test substance or extract EC50 (antioxidative ) in µg/ml Ascorbic acid p.a. 25 CitroZine TM 175 Red wine (Dornfelder) 275 Microalgae & mineral formulation 500 Aronia extract (dry powder) 600 Dandelion extract (fresh) 2,000 Cranberry extract (dry powder) 2,000 Cystus extract (dry powder) 2,750 Rosemary extract (fresh) 3,000 Basil extract (fresh) 3,500 2 Anti-inflammatory potential (energy metabolism of functional neutrophils) As can be seen in Figure 6, CitroZine reduced the energy metabolism of functional neutrophils as inflammation-mediating cells in a dose-dependent manner. In a second set of experiments it was assured that no unwanted interactions between the tetrazolium dye und CitroZine were observed (Figure 6). As a matter of fact, the range of test concentrations

9 - 9 - was limited to a maximum concentration of 250 µg/ml (= 0.025%). Since cell vitality was reduced at concentrations 250 µg/ml (Figure 7), but the energy metabolism was reduced even at lower concentrations in a dose-dependent manner, it is concluded that the ingredients of CitroZine possess a specific anti-inflammatory effect by reducing the energy metabolism of functional neutrophils. The EC50 was calculated from mean V (mod/min) of the initial linear part of the curve to be 150 µg/ml (= vol%). 3 Anti-inflammatory potential (oxidative burst of functional neutrophils) CitroZine inactivated superoxide anion radicals generated by PMA-stimulated functional neutrophils in a dose-dependent manner (Figures 8 and 9) at concentrations which was demonstrated not to cause a loss in cell vitality (Figure 7). The EC50 for anti-inflammatory potential was calculated from the mean V (mod/min) of the part of the curve within the time period of 20 to 60 min to be 130 µg/ml (= vol%). Comparative anti-inflammatory data of reference substances or extracts are given in Table 3 demonstrating that CitroZine was the best natural product/mixture tested in this respect. Table 3: Comparative anti-inflammatory values for various test substances or extracts as obtained under similar test conditions as described here. Ibuprofen sodium salt is synthetically produced (CAS No ) and was used in analytical grade (p.a.) purity. Test substance or extract EC50 (anti-inflammatory ) in µg/ml Ibuprofen sodium salt p.a. 50 CitroZine TM Red wine (Dornfelder) Bilberry extract (dry powder) 1,000 Dandelion extract (fresh leaves) 2,500 Cranberry extract (dry powder) 2,500 Aronia extract (dry powder) 5,000 Coltsfoot extract (fresh leaves) 10,000 Internal annotation: Antioxidant data are based on experiments and ; anti-inflammatory data on experiments (energy metabolism & interaction dye-test subst. and (oxidative burst).

10 Optical density [OD nm] control 10 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 250 µg/ml 500 µg/ml 1,000 µg/ml 2,500 µg/ml 5,000 µg/ml Optical density [OD nm] control 10 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 250 µg/ml 500 µg/ml 1,000 µg/ml 2,500 µg/ml 5,000 µg/ml 0.0 Figure 3: Presentation of two experiments with CitroZine showing the increase in optical density with incubation time due to the cleavage of the dye by free superoxide anion radicals. Note the dose-dependent inactivation of free radicals in this cell-free test assay. 0.0 Optical density [OD nm] control 10 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 250 µg/ml 500 µg/ml 1,000 µg/ml 2,500 µg/ml 5,000 µg/ml Figure 4: Presentation of the interaction between CitroZine and the tetrazolium dye used in the assays. As can be seen, concentrations > 500 µg/ml (= 0.05%) cause changes in optical density, thus interfering with measurement data. Therefore, a maximum concentration of 500 µg/ml is concluded to be interaction-free and the measurement data up to that concentration reflect the real situation.

11 Inactivation of free superoxide radicals in %. (complete inactivation = 100%) Concentration of test substance in µg/ml Figure 5: Inactivation of free superoxide anion radicals by CitroZine. Data are calculated from mean V (mod/min) of the initial linear part of the curves as depicted in Figure 3. Only test concentrations up to 500 µg/ml (= 0.05%) are taken into account. Note that radical inactivation becomes statistically significant (p<0.01; Student s t-test) at 100 µg/ml (= 0.01%) and that higher concentrations cause a nearly complete inactivation of free superoxide anion radicals. Data represent mean value ± standard deviation.

12 - 12-4,0 control 10 µg/ml 50 µg/ml 0.05 control 10 µg/ml 50 µg/ml 3,5 100 µg/ml 250 µg/ml 100 µg/ml 250 µg/ml Optical density [OD nm] 3,0 2,5 2,0 1,5 1,0 Optical density [OD nm] ,5 0, Figure 6: Dose-dependent reduction of energy metabolism of unstimulated functional neutrophils (left). From this measurement it cannot be decided whether this reduction is caused by successive acidification and loss of cell vitality or by a specific inhibition of CitroZine ingredients. The right part demonstrates that unwanted interactions between tetrazolium dye and test substance were not observed under these test conditions. Cell vitality in % vs. control (control = 100%) Concentration of test substance in µg/ml Figure 7: Dose-dependent reduction of cell vitality at concentrations 250 µg/ml (= vol%) as checked by mean V (mod/min) for 30 min after exposure to CitroZine for the same time period as in the assay for energy metabolism (180 min).

13 PMA-induced oxidative burst [OD nm] control 5 µg/ml 10 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 250 µg/ml 500 µg/ml Inactivation of superoxide anion radicals [%].. (complete inactivation = 100%) µg/ml 10 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 250 µg/ml 500 µg/ml PMA-induced oxidative burst [OD nm] control 5 µg/ml 10 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 250 µg/ml 500 µg/ml Inactivation of superoxide anion radicals [%].. (complete inactivation = 100%) µg/ml 10 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 250 µg/ml 500 µg/ml PMA-induced oxidative burst [OD nm] control 5 µg/ml 10 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 250 µg/ml 500 µg/ml Inactivation of superoxide anion radicals [%].. (complete inactivation = 100%) µg/ml 10 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 250 µg/ml 500 µg/ml Figure 8: Dose-dependent inactivation of superoxide anion radicals by CitroZine as observed in three different experiments. Radicals were generated by PMA-stimulated functional neutrophils. The left column represents the absolute measurement values and the right column the relative inactivation of superoxide anion radicals in comparison to untreated control.

14 Inactivation of superoxide anion radicals [%]. (complete inactivation = 100%) Complete inactivation Concentration in µg/ml Figure 9: Dose-dependent inactivation of superoxide anion radicals by CitroZine. Radicals were generated by PMA-stimulated functional neutrophils Data are calculated from the mean V (mod/min) of the final linear part of the curves (20-60 min) as depicted in Figure 8. Only test concentrations up to 500 µg/ml (= 0.05%) are taken into account. Note that radical inactivation becomes statistically significant (p<0.01; Student s t-test) at 100 µg/ml (= 0.01%) and that higher concentrations cause a nearly complete inactivation of free superoxide anion radicals. Data represent mean value ± standard deviation.