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Anxiety and disorders associated with anxiety are increasing rapidly throughout the world. In the present research Arnica montana (Asteraceae) was studied on neuro-pharmacological and analgesic effect in mice. Decreased locomotor and exploratory activities (open field, head dip cage, stationary rod, cage cross, forced swimming and light and dark) were observed in mice at the dose of 100 mg/kg. These results were found significant at P<0.05 when compared with Diazepam 2mg/kg. A. montana crude extract also exhibited potent analgesic effect at the doses of 50 and 100 mg/kg (P<0.05). Aspirin 300 mg/kg was used as a reference drug. Highest % of inhibition (57.6%) was observed in the third phase of acetic acid induced writhing test at the dose of 100 mg/kg of A. montana. Whereas, significant analgesic response was also observed at the low dose (50mg/kg). The percentage inhibition by formalin induced licking biting was found significant in the 2nd (76.1%) and 3rd (19.8%) phase at 50 mg/kg. These results were suggested that the extract of A. montana possess significant analgesic and anxiolytic effect.
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Research Article
NEURO-PHARMACOLOGICAL AND ANALGESIC EFFECTS OF ARNICA MONTANA EXTRACT
MANSOOR AHMAD*1, FARAH SAEED2, MEHJABEEN3, NOOR JAHAN4
1Research Institute of Pharmaceutical Sciences, University of Karachi, 2Department of Pharmacognosy, Dow College of Pharmacy, Dow
University of Health Sciences, 3Department of Pharmacology, Faculty of Pharmacy, Federal Urdu University of Arts Science and
Technology, 4Department of Pharmacology, Dow College of Pharmacy, Dow University of Health Sciences.
Email: herbalist53@yahoo.com, mehjbn1@gmail.com
Received: 05 Aug 2013, Revised and Accepted: 07 Sep 2013
ABSTRACT
Anxiety and disorders associated with anxiety are increasing rapidly throughout the world . In the present research Arnica montana (Asteraceae)
was studied on neuro-pharmacological and analgesic effect in mice. Decreased locomotor and exploratory activities (open field, head dip cage,
stationary rod, cage cross, forced swimming and light and dark) were observed in mice at the dose of 100 mg/kg. These results were found
significant at P<0.05 when compared with Diazepam 2mg/kg. A. montana crude extract also exhibited potent analgesic effect at the doses of 50 and
100 mg/kg (P<0.05). Aspirin 300 mg/kg was used as a reference drug. Highest % of inhibition (57.6%) was observed in the third phase of acetic
acid induced writhing test at the dose of 100 mg/kg of A. montana. Whereas, significant analgesic response was also observed at the low dose
(50mg/kg). The percentage inhibition by formalin induced licking biting was found significant in the 2nd (76.1%) and 3rd (19.8%) phase at 50
mg/kg. These results were suggested that the extract of A. montana possess significant analgesic and anxiolytic effect.
Keywords: Arnica montana, Anxiolytic, Neuro-pharmacological activities, Diazepam, Analgesic.
INTRODUCTION
Anxiety and its related disorders have become a global problem and
affected most of the population world-wide [1]. Benzodiazepines are
commonly used for the treatment of anxiety [2-3]. They have
pronounced anxiolytic effect along with numerous unpleasant side-
effects. The low safety profile of benzodiazepines initiated the
researchers to explore natural world and discover new compounds
with lesser adverse effects [4-5].
Arnica montana (Wolf's Bane, Leopard's bane), belongs to family
Asteraceae. It contains volatile oil, carotenoids, flavonoids, tannins,
resins and triterpenic alcohol. A. montana, has antiseptic, anti-
inflammatory, anti-bacterial, decongestive and anti-fungal
properties. It also stimulates the forming the granular tissues and
thus accelerating the healing process [6-9].
The arnica flowers are used to treat the pale face skin complexion,
wounds, bruises and burns. The treatment of dislocations, bacterial
infections, skin cancer, bronchitis, tonsillitis, pharyngitis, flu, lung
cirhosis, cystitis, nephritis, kidney infections, coronary
insufficiencies, hypertension, angina, cerebral trauma, headaches,
paresis, semi-paresis, insomnia, heart palpitations, nightmares, night
terrors, moral depressions, neurosis, hysteria etc are the other uses
of A. montana [10-12].
MATERIAL AND METHOD
Arnica montana plant extract was purchased from Bioron suppliers.
The extracts obtained were stored in cool, dry place for further
studies. Male albino mice weighing 20-25g were maintained under
standard nutritional and environmental conditions throughout the
experiment. The animals had access to water and food ad libitum.
The animals were deprived of food 12 h before experimentation.
Diazepam (2 mg), aspirin (300 mg), formaldehyde and acetic acid
were purchased from the local Pharmacy and retail chemical stores.
All drugs and A. montana extract were dissolved in saline solution
which used as a vehicle to a desired concentration. Then, they were
filtered through gauze and given to the animals via the intra-gastric
feeding tube. All administered substances including the A. montana
suspension were freshly prepared.
Neuro-pharmacological activities were studied by open field,
traction, head dip, rearing test and swimming induced depression
test. All tests were performed in a calm and peaceful environment.
Animals were divided in to 5 groups (n= 6 in each group). Group I:
control group mice, Group II: Positive control treated group. In each
test, the positive control group was treated with the standard
reference drugs. In order to determine anxiolytic effect, the animals
were treated with diazepam (2 mg kg-1). Group III- V: The mice
treated with different doses of the test extract respectively
(500mg/kg, 300mg/kg, 100 mg/kg), via oral route [13].
Neuro-pharmacological activities
Open field test
Open-field test is a rodent model used for measuring anxiety and
exploration as well as locomotion in mice. Open field test area is
made of plastic walls and floor divided into nine square of equal
area. It is used to evaluate the exploratory activity of animal. The
observed parameters in this study are the number of square crossed
in 30 minutes [14].
Light dark test
Light and dark test is one of the apparatus designed to test anxiolytic
behavior in mice. The apparatus consists of a plastic box with two
compartments one of which is made of transparent plastic and the
other of black colour plastic. Each animal is placed at the center of
the transparent compartment and then the number of entries in
each space, as well as, time spent in each compartment is recorded
for 30 minutes [15-16].
Head-dip test
This study was conducted using wooded apparatus measuring
40×40 cm with 16 evenly spaced holes. Thirty minutes after
treatment, the mice were placed singly on a board with 16 evenly
spaced holes and the number of times the mice dipped their heads
into the holes was noted. The control and drug treated animals were
placed individually in the head dip box and the observations were
made for 30 minutes [17].
Cage crossing movement
The cage crossing test was performed in a specifically designed mice
cage having rectangular shape. Both control and treated mice were
placed separately in the cage and their movements were noted for
30 minutes [18].
Forced swimming test
In this test mice are forced to swim in a restricted space from which
there is no escape and become immobile. Individual mice were
forced to swim in an open cylindrical container containing 7 cm of
water at 22.0 ±1.0oC; the duration of immobility or struggling in a
period of 6 minutes was recorded. Immobility was evaluated as
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 5, Issue 4, 2013
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Ahmad et al.
Int J Pharm Pharm Sci, Vol 5, Issue 4, 590-593
591
when mice ceased to struggle and remained floating in the water,
making only necessary movements necessary to keep its head above
water. At the end of the session, the animal was removed from water
and dried gently [19-20].
Stationary rod test
The stationary rod test was used to assess learning ability and
locomotor activity. The test apparatus consisted of horizontal
stainless steel rods with the platform. At first the mice were trained
and then they were allowed to balance on stationary rod. During the
observation time mice maintained balance, travelled and fall from
the stationary rod was noted [21].
Analgesic activity (by acetic acid)
The analgesic effect was assessed by acetic acid induced writhing
test in mice. The animals were administered orally with the test
samples and reference standard drug aspirin (300 mg/kg). 0.6%
acetic acid per kg was injected intra-peritoneal. The resultant
abdominal writhes consist of a contraction of body muscles and the
stretching of hind limb. These abdominal contractions were counted
over three periods of 10 min each after injection of acetic acid. The
anti-nociceptive activity was considered as the reduction of these
writhes in the treated animals. The % inhibition of the test samples
was calculated by using the following formula: (number of writhing
of control) (no of writhing of test group)/ (number of writhing of
control) ×100 [22].
Analgesic activity (by Formalin)
In this test, 2.5% formalin solution (0.05 ml) was injected in the sub-
plantar region of right hind paw of the mice to induce pain. Aspirin
was administered (30 min before formalin injection) orally to serve as
a control. The A. montana extracts (50 and 100 mg/kg) were
administered orally 60 min before the reaction. The animals were
placed in transparent cage for recording observations and the time
spent in licking and biting responses of the injected paw was taken as
indicator of pain response. Responses were measured for 30 min after
formalin injection. The reduction in the number of licking and biting
responses is the inhibition of the pain score and the % inhibition can
be calculated by the following expression: (mean of control group)
(mean of treated group)/ (mean of control group) ×100 [23].
Statistical analysis
The results were expressed as mean ± S.E.M. All statistical
comparisons were made by means of Student’s t-test and a P value
smaller than 0.05 was regarded as significant [24].
RESULTS
Locomotor and exploratory behavior of mice
The anxiolytic activity was assessed using open field, head dip and
stationary rod apparatus. The most significant CNS depression
effect was observed at the dose of 100 mg/kg of A. montana
extract. In open field activity the results were found to be
11.331.73 counts in 30 minutes. In head dip test, the mice dipped
head 242.65 times. Number of entries in light compartment is
5.51.51 times. The readings of cage cross is 24.672.41 times. In
forced swimming test (FST) the Mean forced mobility time was
1.780.12 seconds. Mean time of mobility on stationary rod was
12.50.83 seconds. Locomotor and exploratory activity was
reduced in comparison to control and standard drug Diazepam (2
mg/kg-1) (Table 1 and Graph 1).
Acetic Acid Test
Analgesic activity is widely assessed by the method of acetic acid
induced abdominal constrictions. 0.6% (10 ml/kg i.p) acetic acid
solution was administered in mice and the abdominal constrictions
(writhes) were observed after 05 minutes. The writhes were
counted for three phases, each of 10 minutes respectively. The
inhibition of acetic acid induced writhes by Aspirin was as follows;
1st; 66.7%, 2nd; 32.2%, 3rd phase; 35.5%. Where as A. montana, at the
dose of 100 mg/kg exhibited maximum inhibition (57.6 %) in 3rd
phase. (Table 2 and Graph 2).
Formalin Test
The results exhibited prominent analgesic effect in comparison with
aspirin. The analgesic effect of A. montana showed maximum
inhibition of the licking and biting response (76.1%) induced by
formalin at the dose of 50 mg/kg in 2nd phase. Aspirin in 1st, 2nd and
3rd phase showed was 21.7%, 84.3% and 22.8% inhibition
respectively (Table 3 and Graph 3).
Table 1: Neuro-pharmacological activities of Arnica montana
Treatment
Open field
Head Dip cage
Light and dark
Cage cross
Stationary rod test
Control
209.833.97
109.53.67
17.51.99
68.333.68
171.05
A .montana 500mg/kg
1257.94
65.334.19
15.831.55
26.334.46
22.161.27
A. montana 300mg/kg
187.672.98
67.832.90
212.28
97.832.76
18.161.03
A. montana
100mg/kg
11.331.73
242.65
5.51.51
24.672.41
12.50.83
Standard-Diazepam 2mg/kg
12.50.83
11.50.83
10.4
19.50.83
11.331.73
(fall)
Results were expressed along mean standard error mean. Significance of data were evaluated at P 0.05 student t test
Graph 1: Neuro-pharmacological activities of A. montana
Ahmad et al.
Int J Pharm Pharm Sci, Vol 5, Issue 4, 590-593
592
Table 2: Analgesic activity of A. montana (by acetic acid)
Treatment
Dose mg/kg
Phase 1
Phase 2
Phase 3
Mean No. of Writhes
% of
Inhibition
Mean No. of Writhes
% of
Inhibition
Mean No. of Writhes
% of
Inhibition
Control
0.5 ml saline
19.5±0.83
-
15.5±0.83
-
14.16±1.03
-
Aspirin (300mg)
6.5±0.83**
66.7
10.5±1.08
32.2
9.16±0.77
35.3
A. Montana (100 mg)
8.33±1.43*
57.4
10±1.41*
35.4
6±0.56**
57.6
A. Montana (50 mg)
10.67±0.96
45.1
8.83±0.86**
43
6.67±0.73*
52.8
Results were expressed along mean standard error mean. Significance of data were evaluated at P 0.05 student t test
Graph 2 Analgesic Activity of A. montana (by Acetic Acid)
Table 3: Analgesic activity of A. montana (by formalin)
Treatment
Dose mg/kg
Phase 1
Phase 2
Phase3
Mean No. of biting
& licking
% of Inhibition
Mean No. of
biting & licking
% of Inhibition
Mean No. of
biting & licking
% of Inhibition
Control
0.5 ml saline
19.16±1.03
-
10.67±0.54
-
5.83±1.03
-
Aspirin(300mg)
23.33±1.15
21.7
1.67±0.61
84.3
4.5±0.83**
22.8
A. montana (100 mg)
13.16±3.06**
45.5
1.16±0.52
73.1
6.16±1.86
5.6
A. montana(50 mg)
19±3.42
0.83
0.16±0.18**
76.1
4.67±1.40*
19.8
Results were expressed along mean standard error mean. Significance of data were evaluated at P 0.05 student t test
Graph 3: Analgesic Activity of A. montana (by Formalin)
DISCUSSION
Stress is the causative factor behind anxiety and depression. It is
estimated that by 2020, these disorders will become the second
number cause of disability [25]. In present study the neuro-
pharmacological screening like open field activity, stationary rod
activity test and head dip activity of A. montana were evaluated. The
extract decreased the exploratory and locomotor activities in mice.
Our observations indicated that A. montana treated mice spent more
time in light compartment and therefore, had anxiolytic effect. The
light and dark box test in mice is use to assess anxiolytic activity
[26]. The lesser number of entries in the light box reflects the
sedative and anxiolytic effect of the extract [26].
The development of immobility, during swimming test indicated
that the cessation of affective/motivational behavior. Plants rich in
tannins and flavonoid content have been found to be of therapeutic
significance in treating many CNS disorders [27]. A. montana also
contains tannins and flavonoids and maybe due to the presence of
these constituents it exhibited pronounced anxiolytic effect.
The present study revealed that A. montana possess significant
analgesic effect. The medicinal uses of A. montana are well
established and have been effectively used since ancient time for the
treatment of strains, sprains and bruises. A. montana contains
helenalin, a sesquiterpene lactone that is a major ingredient having
anti-inflammatory effect due to which it is mostly used for the
Ahmad et al.
Int J Pharm Pharm Sci, Vol 5, Issue 4, 590-593
593
treatment of bruises. It can be suggested that analgesic effect may be
due to the presence of volatile oil.
CONCLUSION
Our present study revealed that A. montana possesses significant
analgesic and anxiolytic effect, along with prominent decrease in
neuro-pharmacological activities due to its active chemical
constituents. However, further studies are required to confirm the
mechanism of action behind the effects observed in our study.
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... 3 New synthesized compounds as well as drugs derived from traditional herbs may have a possible therapeutic relevance in the treatment of anxiety. 4 In conclusion, strategic approaches aimed at accelerating promising research directions and enhancing quality standards of ongoing investigations into putative psychotropic agents from natural sources are recommended. 5 Thus it is desirable to explore anxiolytic agents derived from herbs. ...
... 7 Arnica montana at the dose of 100mg/kg is known to possess significant anxiolytic effect. 4 In this study alprazolam is used as standard drug and is having less side effects and Arnica montana not yet compared with alprazolam. Hence the present study is undertaken to compare anxiolytic effects of Arnica montana with standard antianxiety drug alprazolam in rats. ...
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Background: Anxiety affects around 7.3% of the total population worldwide. Benzodiazepines are preferred anxiolytic agents and are still frequently used in spite of the side effect profile including muscle relaxation, memory disturbances, sedation, physical dependence. Arnica montana, a traditional herb is known to possess significant anxiolytic effect at the dose of 100mg/kg. In this study, Arnica montana has been compared for the first time with alprazolam, a most commonly used anxiolytic drug.Methods: Forced swim test was used to induce anxiety. Anxiolytic action of study drugs which were given orally, was evaluated using Open field test (OFT) in healthy wistar rats of either sex. Behavior of rats, locomotion and number of squares crossed was recorded. Rats were divided into four groups with eight rats in each group. Study groups were Group I Control; Group II Alprazolam 0.08mg/kg; Group III Arnica montana extract (AME) 100mg/kg; Group IV AME + Alprazolam group 100mg/kg+0.08mg/kg. Statistical analysis was done using ANOVA followed by Tukey’s test (p<0.05).Results: Increase in frequency of rearing was significant (p<0.05) in AME group and highly significant (p<0.001) in Alprazolam and combination group in comparison to control. Decrease in frequency of grooming was highly significant (p<0.001) in Alprazolam and combination group. AME also showed significant (p<0.05) decrease in grooming activity.Conclusions: Arnica montana extract showed anxiolytic activity and can be used as an add on drug after further studies and validation in the treatment of anxiety disorders.
... The development of immobility during forced swimming test indicated the cessation of affective/motivational behaviour. [50] Antioxidant and protective effect Arnica montana extract shows significant antioxidant potential. DPPH (2,2 0 -diphenyl-1-picrylhydrazyl radical) free radical scavenging method and phosphomolybdate method have been utilized to determine the antioxidant potential of the plant. ...
... In this study, methanol was used as control and permethrin as standard. [50] Hypopigmentation activity Skin withers with age and also by different environmental stressors such as solar ultraviolet radiation, which leads to skin damage such as thickening like plaque, deep furrowing, wrinkle formation, erythema, loss of skin tone and also hyperpigmentation as a result of increased melanin formation. AM-2 (helenalin 2-methylbutyrate) or A. montana extract when used to treat cultured mouse melanoma cells boosts the HSP70 gene expression with increase in dose and also activates the transcription factor for hsp genes, i.e. heat shock factor-1. ...
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... Plants used for their analgesic effects include arnica, boneset, comfrey, mullein, potatoes, and white oak. The analgesic effects of arnica have been described (Ahmad, 2013), and randomized doubleblind trials revealed that preparations of this plant exhibited a powerful analgesic effect in patients suffering from osteoarthritis but did not mitigate after-surgery pain (Knuesel et al., 2002;Stevinson et al., 2003;Widrig et al., 2007). Mullein (Verbascum sp.) is composed of several bioactive compounds including the phenylethanoid glycoside verbascoside, and a Verbascum extract and verbascoside were found to have analgesic properties in a carrageenan-induced hind paw edema mouse model (Akdemir et al., 2011). ...
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Ethnopharmacological relevance: Parkinson's disease (PD) is a multifactorial neurodegenerative disorder affecting 5% of the population over the age of 85 years. Current treatments primarily involve dopamine replacement therapy, which leads to temporary relief of motor symptoms but fails to slow the underlying neurodegeneration. Thus, there is a need for safe PD therapies with neuroprotective activity. In this study, we analyzed contemporary herbal medicinal practices used by members of the Pikuni-Blackfeet tribe from Western Montana to treat PD-related symptoms, in an effort to identify medicinal plants that are affordable to traditional communities and accessible to larger populations. Aim of the study: The aims of this study were to (i) identify medicinal plants used by the Pikuni-Blackfeet tribe to treat individuals with symptoms related to PD or other CNS disorders, and (ii) characterize a subset of the identified plants in terms of antioxidant and neuroprotective activities in cellular models of PD. Materials and methods: Interviews of healers and local people were carried out on the Blackfeet Indian reservation. Plant samples were collected, and water extracts were produced for subsequent analysis. A subset of botanical extracts was tested for the ability to induce activation of the Nrf2-mediated transcriptional response and to protect against neurotoxicity elicited by the PD-related toxins rotenone and paraquat. Results: The ethnopharmacological interviews resulted in the documentation of 26 medicinal plants used to treat various ailments and diseases, including symptoms related to PD. Seven botanical extracts (out of a total of 10 extracts tested) showed activation of Nrf2-mediated transcriptional activity in primary cortical astrocytes. Extracts prepared from Allium sativum cloves, Trifolium pratense flowers, and Amelanchier arborea berries exhibited neuroprotective activity against toxicity elicited by rotenone, whereas only the extracts prepared from Allium sativum and Amelanchier arborea alleviated PQ-induced dopaminergic cell death. Conclusions: Our findings highlight the potential clinical utility of plants used for medicinal purposes over generations by the Pikuni-Blackfeet people, and they shed light on mechanisms by which the plant extracts could slow neurodegeneration in PD.
... Acidic polysaccharides derived from an extract of A. montana have revealed in invitro studies immune-stimulating activities. The constituents of A. montana possess significant analgesic and anxiolytic effect 4 . A. montana can increase blood pressure; therefore, it should be used with caution in patients suffering from hypertension, heart disease or circulatory disorders 5,6 . ...
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Arnica montana Linn. (Asteraceae) is well known for curing mental, emotional, spiritual shocks and physical injuries. Our present research is carried out to investigate the toxicological effect on albino rabbits of low dose of Arnica montana when administered orally for a period of three months. Hematological, biochemical, histo-pathological studies and urine analysis were carried out to evaluate effects of low dose of extract when given for 90 days. In male test group, all the blood parameters were observed to be elevated while in female were found to be lowered except platelet count that was elevated. Creatinine and uric acid levels were found raised in female test group as compared to male test group. Cardiac enzymes were elevated in male test group and were lowered in female test group. Male test group revealed decreased lipid profile and liver enzymes parameters whereas, female test group exhibited raised lipid profile and liver enzymes levels, respectively. Urine volume and specific gravity were found lowered in both male and female test group. Blood cells were observed in urine of female test group. No significant toxic effects were observed with low dose treatment of Arnica montana on the stomach, kidney and liver tissues of male rabbits. Whereas, areas of myocytolysis in right ventricular wall and inter-ventricular septum was observed on the heart tissues. Our results justify the well-documented uses of Arnica montana. © 2016, International Journal of Toxicological and Pharmacological Research. All rights reserved.
... The open field apparatus designed in the laboratory consists of 76 × 76 cm square area with opaque walls 42 cm high. The floor is divided by lines into 25 equal squares (Ahmad et al., 2013). Mice were placed in the center square of the open field (one at a time). ...
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This reference book contains a comprehensive selection of the most frequently used assays for reliably detecting pharmacological effects of potential drugs, including tests for cardiovascular, analgesic, psychotropic, metabolic, endocrine, respiratory, renal, and immunomodulatory activities. Each of the over 700 assays comprises a detailed protocol with the purpose and rationale of the method, a description of the experimental procedure, a critical assessment of the results and their pharmacological and clinical relevance, and pertinent references. Identification of specific tests is facilitated by the enclosed CD-ROM which allows for a quick and full text research. An appendix with guidelines and legal regulations for animal experiments in various countries will help to plan these experiments properly in accordance with the welfare of laboratory animals.
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Arnica preparations are used for the topical treatment of haemotomas, contusions, sprains, rheumatic conditions, superficial inflammations of the skin and for local inflammations in the mouth. In addition topical Arnica preparations, mostly combinations, are used for the treatment of venous conditions in the lower extremities. The botanical and phytotherapeutical aspects of the two officinal species of Arnica, A. montana and A. chamissonis, are discussed in this article as well as the pharmacological effects of the individual ingredients. The use of Arnica preparations, their toxicity and adverse effects as well as the important phytomedicines containing Arnica at present on the market in Germany are presented in an overview. In addition a short description is given of the preparations containing Arnica that are used in other therapeutical categories, such as e.g. homoeopathy, beside Arnicae flos. Although a lot is known about the pharmacological properties of the constituents of Arnica species, the efficacy of Arnica preparations, however, has not yet been demonstrated sufficiently by controlled clinical studies. Nevertheless, the value of Arnica as a phytotherapeutic remains beyond doubt.