This study was carried out on Apis mellifica L., Digitalis purpurea L., Sambucus nigra L., Thuja occidentalis L., Urtica urens L., Arctostaphylos uva-ursi L., with the objective to explore the neuro-pharmacological activities of these drugs which are in current use of cardiac diseases, skin problems (dermatitis), urinary tract infection, gastro-intestinal tract disorders, allergic conditions, maintaining normal blood pressure, in the removal of kidney stone etc. These activities are reported by different researchers (Klass et al., 2002; Navarro et al., 2000; Bisset and Wichtl, 2001; Chang et al., 2000; Chrubasik et al., 2007; Matsuda et al., 1992).

The  homeopathic   mother   tincture  of  drugs  (sealed  packs  from William Schwabe, Germany) were purchased from local supplier (Apis mellifica L. Lot # 0730707; Digitalis purpurea L. Lot # 2010207; Sambucus nigra L. Lot # 0012188808; Thuja occidentalis L. Lot # 7100710; Urtica urens L. Lot # 3030909 and Arctostaphylos uva-ursi L. Lot # 0512208827). To obtain dried, solid extract, the ethyl alcohol was evaporated by keeping open bottles in sterilized condition in lab for 1 to 2 days at room temperature. Later the residue (dry extract) of each drug was used for experiment. The doses of extract were prepared in 0.5 ml distilled water that is, 300 and 500 mg/kg/0.5 ml (Farah-Saeed, 2014)

 

 

Experimental animals

 

Wistar male mice (25 to 30 g) were obtained from Animal House, Dow University of Health Sciences, Karachi, Pakistan. Animals were kept in Animal House, DUHS, Karachi. They were kept in a climate and light controlled room at least 7 days before initiating neuro-pharmacological activities on them and were provided with food and water ad libitum.

 

 

Assessment of neuro-pharmacological activity

 

Neuro-pharmacological activities (field test, head dip test, cage cross, light and dark and forced swimming activities) were carried out in calm and peaceful environment. Each group comprised of 5 animals (n = 5). Diazepam as 2 mg/kg orally was used as standard. The crude drug and the diazepam were diluted in distilled water and administered orally. The control animals were treated orally with same volume of saline as the crude extract. In all the tests, observations were made after 30 to 40 min of oral dose of the test substance.

 

 

Open field activity

 

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). Number of squares crossed with all four paws was counted for 30 min. Activities of control and drug treated mice were monitored in a balanced design to avoid order effect.

 

 

Light and dark test

 

Light and dark test is one of the apparatus designed to test neuro-pharmacological 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 color 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 min (Crawley and Goodwin, 1980).

 

 

Head dip test

 

Head dip box is used to check exploratory behavior of mice. A specially designed square shaped having several holes at bottom were used in this study. The observation was to count the number of head dips by the animal through these holes in specified time (Durcan and Lister, 1988; Hossain and Uma-Devi, 2001; Sultana and Najam, 2012). The control and drug treated animals were placed individually in the head dip box and the observations were made for 30 min.

 

 

Cage crossing movements

 

The test was performed on mice in a specifically designed box having rectangular shape. Both control and treated mice were placed into the cage and their cage crossing movements were noted in 30 min. The test was performed to evaluate motor activity of mice. This test was performed according to the method described by Najam and Anser (2011).

 

 

Forced swimming test

 

Forced swimming test was performed according to Porsolt (1977, 1978). This test determines the muscle and central nervous system (CNS) activity of the crude extract. Mice were placed individually for six minutes in the specially designed plastic cylinder filled with water at room temperature up to the marked level. Mouse when placed in water suddenly starts to  move  its  front  and  hind  paws.

 

The activity time of animal is determined with the help of stopwatch out of total observation time of six minutes.

 

 

Statistical analysis

 

All values were compared with the control and standard drug reading by taking out mean and standard error of mean. Level of significance was determined by student t-test (Alcaraz et al. 1989).

The neuro-pharmacological activity of six crude extracts were assessed using open field, head dip, light and dark, cage cross and forced swimming activities (Table 1).

 

 

Neuro-pharmacological effects of A. mellifica L. on mice

 

The neuro-pharmacological effects were observed at the dose of 100 mg/kg of A. mellifica extract as follows; in open field activity (28 ± 2.84) counts in 30 min were observed, while in head dip test, the mice dipped head (13.33 ± 2.61) times. At the dose of 300 mg/kg the pronounced depressed effects were observed in case of light and dark, cage cross and swimming activities. Number of entries in light compartment is 9.33 ± 2.93 times. The readings of cage cross is (23.33 ± 2.44) times. In forced swimming test (FST) the mean forced mobility time was (1.25 ± 0.04) s. Locomotor and exploratory activity was observed to be considerably reduced in comparison to control and standard Diazepam (2 mg kg^-1) (Figure 1).

 

 

Neuro-pharmacological effects of D. purpurea L. on mice

 

The most significant neuro-pharmacological effect was observed at the dose of 500 mg/kg of D. purpurea extract as follows; in head dip test, the mice dipped head (19.16 ± 4.92) times. Number of entries in light compartment is 4 ± 1.35 times. The readings of cage cross is (6.67 ± 1.48) times. In forced swimming test (FST) the mean forced mobility time was (4.14 ± 0.48) s. At the dose of 300 mg/kg, in case of open field activity (44 ± 4.54) counts in 30 min were observed. Locomotor and exploratory activity was observed to be substantially reduced in comparison to control and standard diazepam (2 mg kg^-1) (Figure 2).

 

 

Neuro-pharmacological effects of S. nigra L. on mice

 

At the dose of 100 mg/kg of S. nigra, CNS inhibitory effects were observed in following activities; in open field activity (75.16 ± 3.68) counts in 30 min, in light  and  dark  activity 3.83 ± 0.65 times in cage cross test, 8.33 ± 1.01 times. Whereas, in forced swimming test (FST) the mean force mobility time recorded was 0.24 ± 0.03 s, at 300 mg/kg dose of S. nigra extract. At 500 mg/kg of S. nigra, the mice dipped its head 7.16 ± 0.77 times. Locomotor and exploratory activity was observed substantively reduced in comparison to control and standard drug, diazepam (2 mg kg^-1) (Figure 3).

 

 

Neuro-pharmacological effects of T. occidentalis L. on mice

 

The most significant neuro-pharmacological effect was observed at the dose of 100 mg/kg of T. occidentalis extract as follows; in open field activity 10 ± 1.41 counts in 30 min were observed. In head dip test, the mice dipped its head 2.83 ± 0.65 times. In light and dark activity, number of entries in light compartment were found 1.16 ± 0.52 times. The observations of cage cross were found 5.67 ± 0.96 times. In forced swimming test (FST) the mean forced mobility time was 0.29 ± 0.01 s. Locomotor and exploratory activities were noticeably reduced in comparison to control and standard drug, diazepam (2 mg kg^-1) (Figure 4).

 

 

Neuro-pharmacological effects of U. urens L. on mice

 

The neuro-pharmacological effects were observed at the dose of 100 mg/kg of U. urens extract in case of open field activity, 85.83 ± 3.53 counts in 30 min and cage cross 67.5 ± 3.15 times activity. At the dose of 300 mg/kg of U. urens; maximum CNS depression was observed in head dip cage, the mice dipped its head 4.67 ± 1.28 times. Number of entries in light compartment, in light and dark activity was 4.83 ± 1.03 times. In forced swimming test (FST) the mean forced  mobility  time  was 1.37 ± 0.02 s. Locomotor and exploratory activity was observed considerably reduced in comparison to control and standard drug, Diazepam (2 mg/kg) (Figure 5).

 

 

Neuro-pharmacological effects of A. uva-ursi L. on mice

 

At the dose of 30 mg/kg of A. uva-ursi during open field activity 177.5 ± 3.17 counts in 30 min were observed. Whereas, number of entries in light compartment were 10 ± 2.11 times, in light and dark activity. In forced swimming test (FST) the mean forced mobility time was 1.22 ± 0.03 s at the dose of 100 mg/kg. On administration of 500 mg/kg of A. uva-ursi, the mice dipped its head 18.83 ± 5.11 times, during head dip activity and the observations on cage cross were found  28.5 ± 6.13.  Neuropharmacological activities were observed to be significantly lowered in comparison to control and standard drug Diazepam (2 mg kg^-1) (Figure 6). The purpose of carrying out neuro-pharmacological studies is to explore the biological basis of the efficacy of crude extracts for the treatment of central nervous system disorders. These studies are mandatory for the formulation of safe and effective drugs. Most of the crude extracts with neuro-pharmacological activity possess quite non-specific action, affecting several different target receptors, ions, channels and transporters, likewise, the currently available most of the neuroactive drugs (Rang et al., 2007). The main neuro-pharmacological effects exhibited by crude extracts were as follows:   reduction    of    anxiety    and   aggression, sedation and induction of sleep as well as reduction of muscle tone and coordination (Argyropoulos et al., 2000).

 

 

The neuro-pharmacological effects were observed at the dose of 100 and 300 mg/kg of A. mellifica extract. Extensive research was carried out on this insect drug by various researchers (Asafova et al., 1986; Ludyanskii, 1994). The pharmacologically active constituents reported are useful in the treatment of different diseases, for example Melittin is reported to influence the central nervous system; Phospholipase A, prevents neuronal cell death caused by prion peptides; Phospholipase B has detoxicating activity; Apamine stimulates the CNS; MCD stimulates CNS; Adolapin inhibits the specific brain enzymes (Shkenderov and Ivanov, 1983; Son et al., 2007; Urtubey, 2005).

 

Pharmacological    effects   of  D. purpurea  have  been reported by Ayuso et al. (1993) and Navarro et al. (1994). Our neuro-pharmacological results are found similar to the previously reported results by various researchers that may be due to the presence of digitoxin and minerals present in it (Benli et al., 2009; Negi et al., 2012).

 

Locomotor and exploratory activities of mice were significantly reduced by administration of S. nigra extract in comparison to control and standard drug that indicates that the extract of S. nigra acts through GABA receptor and this action may occur due to presence of following chemical constituents of S. nigra, that is, zicrin, prunasin, holocalin, potassium nitrate and choline. In general the change in pharmacological action occur in model experiments on introduction of S. nigra extract are reported due to rutin, quercetin, cyanicrin-3-glucoside, cyanidin-3-sumbubioside, sambunigrin, viburnic acid and vitamin A and C (Mahmoudi et al., 2014).

 

Anxiolytic effect of T. occidentalis was explored and the results were found to be similar to the research work carried out previously by Lokesh et al. (2011), Jahan (2010) and Alam (2009). Our research work revealed that T. occidentalis possessed pronounced depressive response in comparison to the other crude extracts, control and standard drug treated mice. Neuro-pharmacological action was observed in mice treated with U. urens extract may be due to the occurrence of butyric acid, acetylcholine and acetophenone (Tita et al., 1993).

 

Neuro-pharmacological activities on mice treated with A. uva-ursi extract showed significant depressive action in comparison to the control group. This response may be contributed by arbutin, methyl arbutin, hydroquinone and galloyl derivative of arbutin or related compounds (Beaux et al., 1999).

The conclusive remark on neuropharmacological activities of six crude drugs is that the chemical composition of each drug is different from each other but all are effective in reducing the hypothalamus (CNS) activity, therefore, a quick change/alteration in system behavior takes place which is neither toxic nor destructive in action. On the basis of these and other reported results it is recommended as a safe and effective drug.

Authors declared no conflict of interest.