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ORIGINAL PAPER

Effect of the oral administration homeopathic

Arnica montana on mitochondrial oxidative

stress

Ronaldo Ant^onio de Camargo, Ellen Dias da Costa and Rosana Catisti*

Fundac¸~ao Hermınio Ometto, Uniararas, Av Maximiliano Baruto 500, 13607-339 Araras, SP, Brazil

Objective: To analyze the effect of homeopathic Arnica on mitochondrial oxidative

stress induced by Ca2+plus inorganic phosphate and/or Fe2+-citrate-mediated lipid

per-oxidation through changes in oxygen consumption rates.

Methods: Mitochondria were isolated by differential centrifugation from the livers of adult male Wistar rats which had been treated with Arnica montana 6cH, 12cH, 30cH or succussed 30% ethanol (control) for 21 days.

Results: In the presence of antimycin-A, electron transport chain inhibitor, as

evi-denced by antimycin-A insensitive O2consumption, Arnica inhibited lipid peroxidation

of mitochondrial membranes. In oxidative stress conditions, in the presence of Ca2+

and inorganic phosphate, animals receiving Arnica 30cH had a significant decrease in

mi-tochondrial O2consumption compared to control animals.

Conclusion: When administrated orally, Arnica 30cH protects against hepatic

mito-chondrial membrane permeabilization induced by Ca2+ and/or Fe2+-citrate-mediated

lipid peroxidation and fragmentation of proteins due to the attack by reactive oxygen

species. Homeopathy (2013) 102, 49e53.

Keywords: Homeopathic remedy; Arnica montana; Oxidative stress; Mitochondria

Introduction

Numerous studies have investigated the pharmacological activity or efficacy of some homeopathic remedies in pre-clinical studies (in vitro and in animals) however, little is known about the action of these drugs. The effectiveness of homeopathic remedies remains controversial.1,2Arnica montana is one of the homeopathic medicines most frequently studied in the clinical trials. Some clinical trials report an effect similar to placebo.3e5Homeopathic dilutions of Arnica have been used for many years to assist in postoperative recovery. Recognizing the importance that the drug, alone or with another drug, exerts in inflammatory processes, many clinical trials have been performed, demonstrating that this intervention

both pre- and post-operatively can assist in recovering from surgery.6e9To test the effectiveness of homeopathic treatment we used isolated cytoplasmic organelles as an animal-based model. Mitochondria are intracellular organ-elles that produce ATP by oxidative phosphorylation. Pa-rameters for evaluating mitochondrial integrity include: i) respiration and phosphorylation10; ii) ability to capture and retain calcium in its interior11,12; iii) protons ejected from the matrix during respiration generating an electric membrane potential.13

This study evaluated the mitochondrial function and ox-idative stress in preparations of isolated liver mitochondria of rats submitted to treatment with homeopathic Arnica compared to control. We examined mechanisms related to mitochondrial permeability and transient oxidative stress promoted by the uptake of Ca2+ions. This was spe-cifically studied on isolated rat liver mitochondria (RLM), in rats treated orally with succussed 30% ethanol or homeopathic Arnica 6cH, 12cH and 30cH, with the aim of detecting possible activity under known conditions of oxidative stress through the measure of mitochondrial res-piration.

*Correspondence: Rosana Catisti, Fundac¸~ao Hermınio Ometto, Uniararas, Av Maximiliano Baruto 500, 13607-339 Araras, SP, Brazil.

E-mail:rosanacatisti@uniararas.br

Received 9 December 2011; revised 22 October 2012; accepted 7 November 2012

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Experimentalmethods

Animal care

All experiments were conducted according to the Na-tional Institutes of Health Guidelines for Humane Treat-ment of Animals and were approved by the local Animal Care and Use Committee (no 006/2010). From 8 week of age, 24 male Wistar rats were maintained on a 12-h light/ 12 h dark cycle, under controlled temperature (22C), and were kept in individual metabolic cages (Beira-Mar, S~ao Paulo, Brazil), with free access to water and food (Nu-vilab CR-1, Nuvital). The animals were randomly divided into four groups and treated daily for 21 days, received orally once daily at 10:00 am, with automatic pipette and disposable pipette tip, 200mL of 30% ethanol (control, succussed, solvent used in composition of the remedy); 200mL of Arnica 6cH; 200 mL of Arnica 12cH or 200mL of Arnica 30cH. The liver is metabolically very active organ, and any stress or handling the animal may cause changes in liver mitochondrial bioenergetics. Thus, we reproduced in the control group all procedures per-formed in the treated group. Body mass was recorded weekly. After day 21 of treatment, the animals were sacri-ficed after 12 h fasting, by cervical dislocation, by person experienced in the procedure.

Homeopathy specific materials

The Arnica 6cH (Reg. 404615-0), Arnica 12cH (Reg. 404615-1), Arnica 30cH (Reg. 404615-2) and 30% Ethanol (Reg. 404866) were purchased from Homeopathic Phar-macy Nova Era, Araras, S~ao Paulo, Brazil, they were pre-pared according to Brazilian Homeopathic Pharmacopoeia II (Hahnemannian multi-vial-method, on 30% ethanol, 100 times manual vertical shaking against a soft pad). All rem-edies were prepared before the experiments, packaged in amber glass bottles and stored at room temperature pro-tected from light.

Experimental procedures

Rectal temperatures were measured using a digital ther-mometer (BD Basic, Becton Dickinson, SP, Brazil). Ani-mals were adapted to rapid comfortable immobilization and measurements. Temperatures were recorded between 09:00 and 10:00 am, once every 7 days.

Isolation of RLM

RLM were isolated by conventional differential centrifu-gation14at 4C from the livers of adults Wistar strain rats fasted overnight. Livers were quickly weighed after sacri-fice, chopped and minced, then homogenized in a medium containing 250 mM sucrose, 1.0 mM EGTA - ethylene gly-col tetra-acetic acid and 10 mM HEPES - hydroxyethylpi-perazine ethanesulfonic acid buffer, pH 7.2. The mitochondrial suspension was washed twice in the same medium containing 0.1 mM EGTA, and the final pellet was diluted in 250 mM sucrose and 10 mM HEPES buffer, pH 7.2. RLM samples were homogenized for protein content determination.

Oxygen uptake measurements

RLM (0.5 mg/mL) were isolated from untreated male rat liver 8 week of age and added to the temperature-controlled (28C) chamber of Clark-type electrode (Oxytherm Sys-tem, Hansatech Instruments, Norfolk, UK) with gentle stir-ring in reaction medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES buffer, pH 7.2; 10 mM CaCl2, 3 mM iP; 5.0 mM succinate, 4.0mM rotenone.

Af-ter 1 min of mitochondrial preincubation were added none or 1.0mL 30% ethanol (EtOH 30%) or 1.0 mL Arnica 6cH or 1.0mL Arnica 12cH or 1.0 mL Arnica 30cH. Oxygen consumption was recorded over 10 min, assuming solubil-ity at 28C was 210 mmol mL 1. The values represent mean SEM (n = 6). Rotenone, succinate, HEPES were purchased from SigmaeAldrich (St. Louis, MO).

Lipid peroxidation measurements

Castilho et al15demonstrated that RLM were permeabi-lized when incubated in the presence of Ca2+and Fe2+ -cit-rate, a free radical generating system. In these conditions, it was found that the mitochondrial membrane permeabiliza-tion was mediated by lipid peroxidapermeabiliza-tion and protein frag-mentation due to reactive oxygen species (ROS). The lipid peroxidation permeabilization process and protein fragmentation were determined by O2consumption

inde-pendent from antimycin-A because this compound inhibits electron transfer in the Complex III on the inner mitochon-drial membrane. The presence of rotenone, an inhibitor of the mitochondrial electron transport chain, in the standard reaction medium prevents ROS formation at Complex I, by endogenous substrates like NADH - nicotinamide adenine dinucleotide, reduced form. This experimental condition has the advantage of eliminating the involvement of respiration, membrane potential and Ca2+cycling across the inner membrane in mitochondrial swelling.16To evalu-ate a possible effect of homeopathic Arnica treatment under this condition, RLM (0.5 mg/mL) isolated from treated rat groups were added to the temperature-controlled (28C) chamber of Clark-type electrode (Oxytherm System, Han-satech Instruments, Norfolk, UK) with gentle stirring and incubated in reaction medium containing 250 mM sucrose, 10 mM HEPES buffer, pH 7.2; 4.0mM rotenone, 0.5 mM antimycin-A and 2.0 mM citrate. After 1 min of mitochon-drial preincubation were added 50 mM de Fe2+. Oxygen consumption was recorded over 10 min, assuming solubil-ity at 28C was 210 mmol mL 1. The results represent averages SEM of measurements from 6 different animals.

Mitochondrial oxidative stress measurements

Mitochondrial membrane permeabilization induced by Ca2+was assessed by a nonspecific increase in membrane permeability. It can also result in an increase of mitochon-drial ROS release.17 The study of inner mitochondrial membrane permeabilization induced by Ca2+can be asso-ciated with nonspecific increase in membrane permeability that stimulates respiratory rates and decreases the coupling between oxygen consumption and oxidative phosphoryla-tion.18In the presence of lower concentrations of calcium

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(10mM CaCl2), the mitochondrial permeability transition

(MPT) can be stimulated by inorganic phosphate (iP)18 and this effect can be inhibited in the presence of Ca2+ che-lator EGTA. To evaluate a possible effect of homeopathic Arnica treatment under this condition, RLM (0.5 mg/mL) isolated from treated rat groups were added to the temperature-controlled (28C) chamber of Clark-type electrode (Oxytherm System, Hansatech Instruments, Nor-folk, UK) with gentle stirring and incubated in reaction me-dium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES buffer, pH 7.2; 1 mM iP; 2 mM succinate, 5mM ro-tenone and 10mM CaCl2. iP (2 mM) was added after 1 min

of mitochondrial preincubation. Oxygen consumption was recorded over 10 min, assuming solubility at 28C was 210mmol mL 1. The results represent averages SEM of measurements from 6 different animals.

Data analysis

Data were analyzed using GraphPad Prism and Origin Software. KruskaleWallis tests were used to compare groups at control. Significance was set at p# 0.05.

Results

Animal characteristics

The body weight gain of animal groups during the treat-ment period is shown inTable 1A. Liver weight is shown in Table 1B. There was no significant differences in body and liver weight gain between treated or control animals, how-ever, the increase of the values showed animal growth. In our experimental conditions, Arnica had no effect on ani-mals’ body and liver mass. To investigate possible physio-logical changes during treatment, rectal body temperatures were measured (not shown). No differences between the treated and control groups were noted at any time point.

Effectin vitro of Arnica addition in isolated mitochondria

Measurements of oxygen consumption in isolated mito-chondrial suspensions from untreated rat liver were per-formed in order to test the possible in vitro effect of homeopathic drugs over this parameter. Table 2 shows RLM oxygen consumption without additions (RLM) was

lower than others values. The addition of 1.0mL of 30% eth-anol, Arnica 6cH, Arnica 12cH or Arnica 30cH, to the me-dium did not produce significant changes in oxygen consumption, although the addition of 30% ethanol may have induced mild uncoupling of mitochondrial respiration, indicated by the small increase in oxygen consumption.

Arnica protects Fe2+citrate-induced lipid peroxidation

The values found inTable 3represent a series of three experiments carried out under the same experimental con-ditions and show a decrease of oxygen consumption in Ar-nica treated animals. It shows significant inhibition of O2

consumption with Fe2+citrate-induced lipid peroxidation in Arnica treated groups and these results suggest that the RLM membranes subjected to homeopathic Arnica treat-ment are more resistant than RLM membranes subjected to lipid peroxidation of liver mitochondria of the group of animals receiving ethanol 30%. In vivo homeopathic Ar-nica treatment protected against Fe2+citrate-induced lipid peroxidation.

Arnica 30cH inhibited mitochondrial oxygen consumption

Mitochondria isolated from RLM were incubated in a standard reaction medium and variations in the oxygen consumption were observed for 10 min. iP (2 mM) was added after 1 min of mitochondrial preincubation. The values shown in Table 4 represent a series of three

Table 1 Effect of Arnica on body and liver weight. Body mass (A) and liver mass (B) were determined as described in Methods. Treatments were 30% ethanol (Control), Arnica 6cH; Arnica 12cH and Arnica 30cH (all 200mL/day). The values represent mean  SEM (n = 6). Time 0 represents the start of treatment. No statistically significant differences were observed

A Body mass (g)

0 Day 7 day 14 Day 21 Day

Control 252.8 23.35 270.0 21.99 285.6 20.96 300.6 18.27 Arnica 6cH 220.8 18.79 244.0 18.81 261.4 16.08 281.1 17.57 Arnica 12cH 253.2 23.76 271.1 21.31 289.2 21.13 305.5 19.80 Arnica 30cH 241.0 23.91 262.6 21.38 278.4 21.38 297.8 18.13 B Liver mass (g) Control 11.12 0.3481 Arnica 6cH 12.349 1.285 Arnica 12cH 10.565 0.3951 Arnica 30cH 9.1685 0.29152

Table 2 Effect in vitro of addition of Arnica to isolated mitochondria from untreated rat liver on mitochondrial oxygen consumption. RLM(0.5 mg/mL) were isolated from untreated rat liver, and added to standard reaction medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES buffer, pH 7.2; 10mM CaCl2, 3 mM iP; 5.0 mM

succinate, 4.0mM rotenone, stirring for 10 min, at 28C, in the absence (RLM) or presence of 1.0mL 30% ethanol (EtOH 30%); Arnica 6cH; Arnica 12cH and Arnica 30cH. The values represent mean SEM (n = 6). No statistically significant differences were observed

O2consumption (nmoles mg 1min 1)

RLM 83.84 5.301

EtOH 30% 95.90 4.671

Arnica 6cH 100.7 2.326

Arnica 12cH 93.34 2.561

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experiments carried out under the same experimental con-ditions and show a decrease of oxygen consumption in Arnica treated animals. These results suggest that Arnica 30cH treated groups animals were less susceptible to the same MPT conditions than the Control group (p < 0.05) and protected against ROS generation.

Discussion

We encountered difficulty in determining the minimum dose to be used in experiments both in vitro and in vivo. In in vitro experiments, we used a dose to assess mitochon-drial function whose concentration of 30% ethanol alone had minimal effect on isolated mitochondria. Without many previous parameters we chose to use 1mL of the drug as a model. There was no significant change in exper-iments with lower doses. Thus, it can be concluded that in vitro mitochondria isolated from rat livers do not un-dergo a change in respiration caused by the addition of ho-meopathic medicine. However, in the in vivo experiments, we chose convenient dosage sizes because no similar ex-periments have been conducted. In our experimental condi-tions we observed that the homeopathic remedy protected the mitochondria from oxidative damage caused by lipid peroxidation.

In succinate-induced mitochondrial respiration experi-ments, we only observed significant effect with Arnica in 30cH dilution. There are literature reports of antioxidant activity of compounds isolated from plants of the Astera-ceae family, which includes Arnica montana.19,20 However, due to the homeopathic preparation, in which dilution is followed by succussion, no molecules of these

compounds are present in Arnica 30cH. A recent report21 concluded that homeopathic treatment decreased the lipid peroxidation in erythrocytes which shows and reduced ox-idative stress, evidenced by return of plasma vitamin C and erythrocyte superoxide dismutase to normal levels, al-though oxidative stress had not been completely overcome as plasma total antioxidant activity remained low after treatment. Our results suggest a possible action of the drug in mitochondria, the organelle responsible for cellular energy production. Additional experiments need to be car-ried to explore if the action of Arnica in homeopathic dilu-tion can be explained by change in mitochondrial activity.

Conclusion

Oral administration of Arnica 30cH, in adult male rats Wistar rats for 21 days protected against mitochondrial he-patic membrane permeabilization induced by Ca2+ and Fe2+-citrate-mediated lipid peroxidation and fragmenta-tion of proteins due to the attack of ROS. This data can con-tribute to the understanding of a possible action of homeopathic medicine in energy metabolism and mito-chondrial oxidative stress.

Acknowledgments

The authors thank Jose Roberto Passarini Junior and Lia M. G. Neves for excellent technical assistance. RC was the recipient of a fellowship from PROPESq/UNIARARAS. None of the authors have a conflict of interest in respect to the present work. The contributions of the authors were as follows: conception, design of experiments, data analysis and drafting: RC; execution RAC, EDDC and RC.

References

1 Bellavite P, Marzotto M, Chirumbolo S, Conforti A. Advances in homeopathy and immunology: a review of clinical research. Front Biosci (Schol Ed) 2011 Jun 1; 3: 1363e1389.

2 Conforti A, Bellavite P, Bertani S, Chiaroti F, Menniti-Ippolito F, Raschetti R. Rat models of acute inflammation: a randomized con-trolled study on the effects of homeopathic remedies. BMC Comple-ment Altern Med 2007, Jan 17; 7: 1.

3 Paris A, Gonnet N, Chaussard C, Belon P, Rocourt F, Saragaglia D, et al. Effect of homeopathy on analgesic intake following knee lig-ament reconstruction: a phase III monocentre randomized placebo controlled study. Br J Clin Pharmacol 2008; 65(2): 180e187. 4 Plezbert JA, Burke JR. Effects of the homeopathic remedy Arnica

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5 Vickers A, Fisher P, Smith C, Wyllie S. Homoeopathic Arnica 30 is ineffective for muscle soreness after long-distance running: a ran-domized, double-blind, placebo-controlled trial. Clin J Pain 1998; 14: 227e231.

6 Seeley BM, Denton AB, Ahn MS, Maas CS. Effect of homeopathic Arnica montana on bruising in face-lifts: results of a randomized, double-blind, placebo-controlled clinical trial. Arch Facial Plast Surg 2006; 8(1): 54e59.

7 Brinkhaus B, Wilkens JM, L€udtke R, Hunger J, Witt CM, Willich SN. Homeopathic arnica therapy in patients receiving knee surgery: results of three randomised double-blind trials. Com-plement Ther Med 2006 Dec; 14(4): 237e246.

Table 3 Effect of Arnica on Fe2+

citrate-induced lipid peroxidation. RLM (0.5 mg/mL) were incubated in reaction medium containing 250 mM sucrose, 10 mM HEPES buffer, pH 7.2; 4.0mM rotenone, 0.5mM antimycin-A and 2.0 mM citrate. After 1 min were added to 50mM Fe2+. The oxygen consumption was recorded over 10 min, at

28C (*p < 0.05, control vs. treated, KruskaleWallis test). The values represent mean SEM (n = 6)

O2consumption (nmoles mg 1min 1)

Control 39.23 6.678

Arnica 6cH 23.51* 8.851

Arnica 12cH 20.81* 10.24

Arnica 30cH 16.02* 9.292

Table 4 Effect of Arnica 30cH on mitochondrial oxygen consumption. RLM (0.5 mg/mL) were added to standard reaction medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES buffer, pH 7.2; 10mM CaCl2, 1 mM iP; 5.0 mM succinate, 4.0mM

rotenone, and the oxygen consumption, followed by 10 min at 28C. iP (2 mM) was added after 1 min of mitochondrial preincubation (*p < 0.05, control vs. treated KruskaleWallis test). The values represent mean SEM (n = 6)

O2consumption (nmoles mg 1min 1)

Control 43.87 6393

Arnica 6cH 48.80 6.180

Arnica 12cH 39.99 5.077

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8 Oberbaum M, Galoyan N, Lerner-Geva L, Singer SR, Grisaru S, Shashar D, et al. The effect of the homeopathic remedies Arnica montana and Bellis perennis on mild postpartum bleedinge a ran-domized, double-blind, placebo-controlled study e preliminary results. Complement Ther Med 2005; 13(2): 87e90.

9 Robertson A, Suryanarayanan R, Banerjee A. Homeopathic Arnica montana for post-tonsillectomy analgesia: a randomised placebo control trial. Homeopathy 2007; 96(1): 17e21.

10 Kennedy EP, Lehninger AL. Oxidation of fatty acids and tricarbox-ylic acid cycle intermediates by isolated rat liver mitochondria. J Biol Chem 1949; 179: 957e967.

11 Nicholls DG. Calcium transport and proton electrochemical poten-tial gradient in mitochondria from guinea-pig cerebral cortex and rat heart. Biochem J 1978; 170: 511e522.

12 Nicholls DG, Akerman KEO. Mitochondrial calcium transport. Bi-ochim Biophys Acta 1982; 683: 57e88.

13 Akerman KEO, Wikstrom MKF. Safranine as a probe of the mito-chondrial membrane potential. FEBS Lett 1976; 68: 191e197. 14 Schneider WC, Hogeboom GH. Intracellular distribution of

en-zymes. V. Further studies on the distribution of cytochrome C in rat liver homogenates. J Biol Chem 1950; 183: 123e128. 15 Castilho RF, Meinicke AR, Alemida AM, Hermes-Lima M,

Vercesi AE. Oxidative damage of mitochondria induced by Fe(II) citrate is potentiated by Ca2+and includes lipid peroxidation and

al-terations in membrane proteins. Arch Biochem Biophys 1994; 308: 158e163.

16 Vercesi AE, Ferraz VL, Macedo DV, Fiskum G. Ca2+-dependent

NAD(P)+-induced alterations of rat liver and hepatoma mitochon-drial membrane permeability. Biochem Biophys Res Commun 1988; 154: 934e941.

17 Gunter TE, Pfeiffer DR. Mechanisms by which mitochondria trans-port calcium. Amer J Physiol 1990; 258: 755e786.

18 Kowaltowski AJ, Castilho RF, Vercesi AE. Opening of the mi-tochondrial permeability transition pore by uncoupling or inor-ganic phosphate in the presence of Ca2+ is dependent on mitochondrial-generated reactive oxygen species. FEBS Lett 1996; 378: 150e152.

19 Haraguchi H, Ishikawa H, Sanchez Y, Ogura T, Kubo Y, Kubo I. Antioxidative constituents in Heterotheca inuloides. Bioorg Med Chem. 1997 May; 5(5): 865e871.

20 Saluk-Juszczak J, Pawlaczyk I, Olas B, et al. The effect of polyphenolic-polysaccharide conjugates from selected medicinal plants of Asteraceae family on the peroxynitrite-induced changes in blood platelet proteins. Int J Biol Macromol 2010 Dec 1; 47(5): 700e705.

21 Pinto S, Rao AV, Rao A. Lipid peroxidation, erythrocyte antioxi-dants and plasma antioxiantioxi-dants in osteoarthritis before and after ho-meopathic treatment. Homeopathy 2008 Oct; 97(4): 185e189.

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