Zanthoxylum armatum is a small xerophyte, tree or shrub having leaflet blades usually with prickles. Leaves are compound, imparipinnate with 3-7 foliolate and pellucid-punctate. Petiole and rachis are winged. Leaflets are sessile, elliptic to ovate-lanceolate with crenate or entire margins. Flowers born axiliary, minute and polygamous. Calyx 6-8 was acute lobed, petals absent. Male flowers had 6-8 stamens with rudimentary ovary. Female flowers were with 1-3 carpels. Ovary was 1-3 locular. Fruit was small drupes with red color, splitting into two when ripe. Seed are rounded and shining black. Zanthoxylum armatum prefers semi shady or no shade for growth. It grows wild in foothills starting from about 800m up to 1500m in Malakand, Swat, Dir, Hazara, Buner, Muree hills and Rawalpindi [1]. It is locally known as Dambara. Its fruits and seeds are edible and used as potherb species. The plant is used for Pneumonia and tick infestation [2]. Young shoots are used as toothbrush and useful for curing gum diseases. Fruit is used for toothache, dyspepsia, as a carminative and stomachache. Seeds are used for condiment and flavouring agent. Wood is used to make walking sticks [3,4]. Powdered fruit is mixed with Mentha sp. and table salt, eaten with boiled egg for chest infection and digestive problems [5]. Recently, we have tested the leaves and fruits of this plant for various pharmacological activities including antipyretic action [6].

Drugs and other chemicals

Acetylcholine (BDH Chemicals, Poole, England), Potassium chloride (E. Merck Germany), Tyrode’s solution (Prepared from its constituents with their respective concentrations (mM) NaCl 136.9, KCl 2.68, MgCl₂ 1.05, NaH₂PO₄ 0.42, NaHCO₃ 11.90, CaCl₂ 1.8, and glucose 5.55 dissolved in 1 litre distilled water).

Essential oil extraction

A Modified Clevenger type apparatus were used for the extraction of essential oil from the leaves of Zanthoxylum armatum through hydro- steam distillation. The leaves were thoroughly washed, cut into small pieces, placed in distillation flask and subjected to hydrosteam distillation for about 4 hours. The steam and vaporized oil were condensed into liquid by a vertical condenser and collected in measuring tube. Being immiscible and lighter than water, the volatile oil separated out as an upper layer. The oil was then separated from water and collected in small bottles, dried with anhydrous sodium sulphate, sealed, labelled and stored in light resistant vials at 4–6ºC for further use [7].

Animals

Local breed rabbits of either sex with weights in the range of 1.0- 1.4 kg were used. The animals were kept for 14 days before starting the experiments at the “Animal House of Department of pharmacy, University of Malakand” under standard conditions mentioned in the “Animals Bye-Laws 2008 of the University of Malakand (Scientific Procedures Issue- 1).” and fed with standard diet and tap water. The animals were kept in fasting condition for 24 hours prior to the experiments; they were only provided with free access to water.

Gas chromatography- Mass spectrometery (GC-MS)

The essential oil from the leaves of Zanthoxylum armatum were analyzed by GC-MS Model QP 2010 plus (Shimadzu) operating in EI mode at 70 ev, equipped with a split-splitless injector (Split ratio, 1:50). Helium was used as carrier gas with flow rate of 1 ml/min. A capillary column (Length: 30 m, id: 0.25 mm, thickness: 0.25 μm, DB-5MS Agilent technologies, USA) treated with 95% dimethyl- and 5% biphenyl poly silphenylene. HPLC grade dichloromethane was used as reagent. The following conditions were maintained during the operation of GC-MS analysis. Inject temperature: 240 ºC, Ion source temperature (EI): 240 ºC, Interface temperature: 240 ºC, Pressure: 80 KPa, GC program time: 46.67 minutes total, Solvent cut time: 2.5 minutes, MS start time: 3 minutes , MS end time: 46 minutes, Acquisition time: scan, M/Z: 40- 500 [8].

Identification of the Components

The identification of the constituents was based on comparison of the retention times (RT) and mass spectra of the samples with those obtained from standards used. Relative percentage of compounds was calculated from the total chromatogram by using computer software [8].

Antispasmodic activity

Rabbit’s jejunum preparations: Experiments on rabbit’s jejunum preparations were carried out as following [9]. Slaughtered animals were dissected to open abdomen, and jejunum portion(s) were extracted and kept in freshly prepared Tyrode’s solution, aerated with carbogen gas (5% Carbon dioxide and Oxygen mixture) to keep them alive and ready for use. Quiescent sub-maximal doses of acetylcholine (0.3 μM) to the tissues were used when needed for keeping the tissue viable and alive [10]. About 1.5 cm length tissue was mounted in 10 ml tissue bath containing Tyrode’s solution and stabilized for 25-30 minutes. All the processes were carried out at 37+ 1ºC with constant aeration and kept under 1 gram pressure. On attaining reproducible response, test samples at the doses of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0, and 10.0 mg/ml were applied to the bath solution [9,11]. The processes were repeated thrice (n=3) and fall in spontaneous activity was observed to be change of the sample tested. For the determination of possible mode of action, the tissue was pretreated with high concentration of KCl (80 mM in final bath solution). KCl cause depolarization and keep the tissue in a position of sustained contraction [12]. The extract was then applied in cumulative manner to obtain a dose dependent curve and relaxation. Intestinal responses data were recorded using Force Transducer (Model No: MLT 0210/A Pan Lab S.I.) attached with Power lab (Model No: 4/25 T) AD Instruments, Australia. Data was recorded at range of 20 mv, low pass 5 Hz Χ 10 gain using input 1, rate 40/S. Results were expressed as% of KCl induced contraction. Chart 5 (AD Instruments) was used to interpret the graph tracings. Student “t” test was used at 95% confidence interval (CI). ‘P’ values less or equal to 0.05 was considered as statistically significant [10].

Anti bacterial activities

Antibacterial activities of the plant were carried out by agar well diffusion method as used in our previous study [13]. Bacterial strains were first cultured on nutrient broth and incubated for 24 hours prior to experiments. Nutrient agar was melted, cooled to 40ºC and poured into sterilized petridishes. Wells were then bored in media using 6mm diameter with the help of sterile metal cork borer and keeping a distance of 24 mm between two adjacent wells. 4-8 hour old bacterial culture was spread on the surface of nutrient agar with the help of sterilized cotton swab. These processes were repeated thrice turning the plate 60º between each streaking. About l00 μl of 3 mg/ ml of respective extract, dissolved in DMSO was then added to the wells. Other wells were supplemented with DMSO and 10 μg Imipenem served as positive and negative controls. The plates were then incubated for 24 hours at 37ºC. The plates were then observed for zones of inhibition. All the experiments were conducted in triplicate.

Anti-fungal activities

Seven days old fungal cultures (PCSIR labs Lahore Pakistan), test samples, sabouraud dextrose agar,Dimethyle sulphoxide(DMSO), screw cap test tubes, micropipettes, autoclave, incubator, standard antibiotic (Miconazole). Twenty four mg of crude extract was dissolved in 1 ml sterile Dimethyl sulfoxide (DMSO) serving as a stock solution. 4 ml Sabouraud dextrose agar (SDA) growth media was transffered to each screw capped tube, under sterile conditions and autoclaved at 121ºC for 15 minutes. These tubes were then allowed to cool to 50ºC and 400 μg/ml test sample was added to non-solidify SDA tubes, which were then allowed to solidify at room temperature. Next each glass tube was inoculated with 4 mm diameter piece of inoculum removed from 7 days old fungal culture, whereas, agar streak was employed in case of non-mycelial growth. Other media supplemented with DMSO and miconazole antibiotic were used as a negative and positive control respectively. The tubes were incubated at 28 ± 1ºC for 7 days. Cultures were observed twice weekly during incubation. Growth in the media was estimated by measuring linear growth (mm) in the in media loaded with sample, DMSO and miconozole respectively and then percentage inhibition of fungal growth was calculated as follows

% Mycelia inhibition = Gn - Gt / Gn x 100

Where, Gn= Mycelial growth in normal, Gt= Mycelial growth in test [14]

Cytotoxicity

The cytotoxic activity of essential oil from the leaves of Zanthoxylum armatum were tested using brine shrimp assay following recommended method used in our previous work [6,15]. About 20 mg of each extract was dissolved in 2 ml of respective solvent and from this solution transfer 5, 50 and 500 μl to vials (3 vials /concentration). This concentration was equivalent to 10, 100 and 1000 μg/ml, respectively. The solvent were allowed to evaporate overnight. 5 ml with seawater solution (38 g/L) were added to each vial. After 36 h of hatching and maturation of larvae as nauplii, 10 larvae were transferred to each vial using a Pasteur pipette. The vials were then placed at room temperature (25-27ºC) under illumination. Other vials were supplemented with brine solution served as positive controls.

Phytotoxicity

The phytotoxic activity essential oil of the leaves of Zanthoxylum armatum were evaluated using Lamna minor as test species following recommended procedure used in our previous work [6,13]. 15 mg of respective extract was dissolved in 15 ml of respective solvent and from this solution transfer 5, 50 and 500 μl to the flask (3 flasks for each concentration). This concentration was equivalent to 10, 100 and 1000 μg/ml respectively. The solvent was allowed to evaporate overnight under sterilized condition in laminar flow. 20 ml of E. medium was added to each flask. Other flasks (3 for each) were supplemented with E. medium and standard drug (Atrazine) served as negative and positive control. To each flask ten plants with 2-3 fronds were transferred and kept all the flasks under about 12 h day light conditions. Plants were observed daily and on each seventh day the numbers of fronds were counted.

Statistical analysis

All the data expressed as mean ± SEM and the median effective concentrations (EC₅₀) values are given as geometric mean with 95% confidence intervals (CI). One-way Analysis of Variance (one-way ANOVA) followed by Tukey’s post-test was used to determine the significant difference in various doses, P values < 0.05 (P < 0.05) were considered statistically significant. All the graphs, calculation and statistical analyses were performed using GraphPad Prism software version 4.00 for Windows, (GraphPad Software, San Diego California USA, http:// www.graphpad.com).

GC-MS Analysis of essential oil of Zanthoxylum armatum leaves

Essential oils from Zanthoxylum armatum leaf (ZVO) were hydro distillated and GC-MS analysis was carried out for identification of various components (Figure 1). The components with their respective terpenoid nature, percent concentration, retention time and charge to mass ratio are presented in Table 1. Out of the total 34 identified components, monoterpenes alcohol was the largest component (56.57%), followed by monoterpenes hydrocarbins (14.99%), Bergamot mint oil (12.73%), monoterpenes oxygenated (11.39%), monoterpenes acetate (1.9%), monoterpenes aldehyde (0.05%), and sesqueterpenes (2.67%). Among the monoterpene alcohol, beta- Linalool (53.05%) was the major constituents. Other significant components detected in ZVO were monoterpenes of hydrocarbon including alpha-Limonene diepoxide (11.39), alpha- pinene (4.08%), beta- Myrcene (3.69%) and D. limonene (3.1%). α-pinene has strong bactericidal and bacteriostatic potentials. As ZVO containing good amount of α-pinene, it might be used as a good antibacterial as well as bacteriocidal in various formulations. Minor components detected were the monoterpenes including p-meth-1-en-8-ol (2.47%), Geraniol acetate (1.32%), cisbeta- Ocimene (1.29%), Sabinene (1.24%) and p-meth-1-en-8-ol, acetate (1.05%) and sesqueterpenes i.e. Carryophyline (1.32%) and Germacrene- D (1.01%).

Table 1: GC-MS profile of essential oils of the leaves of Zanthoxylum armatum (ZVO).

Figure 1: Typical GC-MS chromatogram of Zanthoxylum armatum leaf essential oil (ZVO) showing the separation of chemical components.

Antispasmodic effect

In the Present study, Essential oil of leaf (ZVO) of Z. armatum was evaluated on the isolated rabbit jejunum for possible antidiarrheal effect, which may be a cheaper and accessible source for treatment of diarrhea and will also provide a scientific proof for its ethno-pharmacological use as an antispasmodic drug. All the samples were tested against spontaneous and potassium chloride induced contracted smooth muscle of the isolated rabbit jejunum. The results of this all bioassays are presented in Figure 2 and 3. To determine the possible mode of action, the tissue was pre-treated with high concentration of KCl (80 mM in final bath solution). KCl cause depolarization and keep the tissue in a position of sustained contraction [16]. The test samples were then applied in cumulative manner to obtain a dose dependant curve and relaxation results were expressed as% of KCl induced contraction [16]. ZVO was also tested against spontaneous and Potassium chloride induced contracted smooth muscle of isolated rabbit jejunum. As compared to control, ZVO significantly relaxed the contracted smooth muscles in both the cases. The spasmolytic effect of the oils started from 0.03 mg/ml and showed 100% effect at 10 mg/ml dose. EC₅₀ values for both spontaneous and KCl induced contraction for ZVO was found 0.22 and 0.73 mg/ml, respectively.

Figure 2: Inhibitory effect of ZVO on spontaneous and high K⁺ induced contractions in isolated rabbit jejunum preparations ^###P < 0.001; shows a comparison of concentration-dependent effects (specified effect compared with the effect of preceding dose) on spontaneous contractions, whereas **P < 0.01; ***P < 0.001; show comparisons of relaxant effects on K⁺ (80 mM)-induced contractions.

Figure 3: Typical tracing showing inhibitory effect of crude extract of ZVO on the spontaneous contractions in isolated rabbit jejunum preparations

The contraction of smooth muscle of rabbit jejunum is due to increase concentration of the free calcium in cytoplasm, which stimulates the chemical mediators which are responsible for contraction. This increase in calcium level may be either due to influx via voltage dependent calcium channels or direct release of calcium from endoplasmic reticulum (calcium store). Thus a periodic depolarization is created due to high speed action potential. When there is increase potassium concentration, the contraction of the smooth muscle will increase due to rapid action potential. When the calcium channel is blocked through calcium channel blocker agents, the contracted smooth muscle will relax [17]. In the present study, the extracts relaxed the contracted muscle, suggesting that the possible mode of action of this plant is either blocking the release of stored calcium from the sarcoplamic reticulum or blocking the calcium channel.

Antibacterial effect

Present study was also carried out for investigation of Z. armatum for such antimicrobial agents. Leaf essential oil of Z. armatum were tested against various gram positive and gram negative bacteria i.e. Micrococcus leutus, Escherichia coli, Staphylococcus aureus, Pasteurella multocida, Pseudomonas aeruginosa, Bacillus subtilis, and Streptococcus viridines as shown in Table 2. The antibacterial actions of the extracts were compared with ciprofloxacin as standard drug. It was observed that ZVO has inhibitory effect against various gram positive and gram negative bacterial strains but the antibacterial potential was greatly varied among the extracts.

Table 2: Antibacterial effect of the essential oil of the leaves of Zanthoxylum armatum.

Antibacterial potential of ZVO was evaluated in the present study and it was found effective against all tested bacterial strains. B. subtilis, and S. viridans were found more susceptible as compared to other strain tested. The maximum effect was observed against M. leutus followed by Streptococcus viridans (17.67 ± 0.58 mm) and B. subtilis (15.83 ± 0.41mm). S. viridans and S. aureus which were found resistant against the other samples tested, affected significantly by ZVO. Essential oils from other plats like Cinnamon, Clove, Geranium, Lemon, Lime, Orange and Rosemary oils were found inhibitory to gram-negative bacteria (E. coli, P. aeruginosa, P. vulgaris) and gram-positive bacteria (B. subtilis and S.aureus). Citrus peel oils have strong antimicrobial activity against various bacterial strains.

Minimum inhibitory concentration (MIC) values were also determined for the test samples. ZVO appeared to be most potent antibacterial agent, as it was effective in inhibiting growth of all bacterial strains with very low MIC values ranging from 0.65- 1.25 μg/ ml with the lowest value for M. leutus and S. viridians (Table 3). MIC values acquired in the present study were very encouraging and further research will enhance opportunity for exploiting ZVO as a strong antibacterial agent, especially the volatile oil for treating halitosis (bad breath) caused by excessive growth of bacteria (Yaegak and Coil, 1999), urinary tract infection caused by E. coli and P. aeruginosai and skin infection caused by M. luteus and B.subtilis. These results are also found very promising as these extract especially the essential oil were found efficient in quite lower concentration.

Table 3: MIC value of the leaf essential oil of Zanthoxylum armatum.

Antifungal effect

In the present study leaf essential oils were evaluated for antimycotic potential against various fungal strains like Trichophyton longifusis, Candida albicans, Fusarium solani, Microsporum canis, Aspergillus flavus and Candida glabrataas. The results are shown in Table 4. A general trend of dose dependency was observed i.e. effect became more pronounced with increasing concentration of the various tested samples.

Table 4: Antifungal effect of the essential oil of leaves of Zanthoxylum armatum.

Antifungal activity of ZVO as a percent inhibition of mycelia growth showed a concentration dependent effect against all the fungi. Best anti-fungal effect was observed against C. albicans (66.67 ± 0.57) followed by A. flauus (55.33 ± 0.57) and F. solani (46.33 ± 0.33). Other fungal strains were also affected at 125 μg/ml concentration. Overall results showed that all fungal strains were inhibited by the ZVO.

Cytotoxicity effect

Brine Shrimp Toxicity bioassay is preliminary study for the detection and development of anti-cancer drugs. Brine shrimp lethality tests were carried out to investigate preliminary cytotoxic potential of the crude ethanolic and n-hexane extract of leaf, bark, fruit and leaf essential oil of Z. armatumi. ZVO showed outstanding mortality rate (100%) at a dose of 1000 μg/ml with 15.90 LC₅₀ values (Table 5). It is needed to carry further detailed investigations for identification and quantification of pharmacologically bioactive specific constituents from Z. armatum.

Table 5: Cytotoxic and Phytotoxic effect of essential oils of the leaves of Zanthoxylum armatum.

Phytotoxicity effect

All the parts showed significant dose dependant phyto inhibition (Table 5). The ZVO showed moderate inhibition at higher dose having FI₅₀ of 2.30 μg/ml. These results suggested that all parts have some active principles with phytotoxic potential and Z.armatum can be a good herbicides or weedicides. Further study is needed to exploit its phytotoxic mechanism and also to identify and quantify the bioactive constituents. It might be helpful to investigate its efficacy in detail as a weeds, pests and disease control agent.

The findings of this research work suggest that the ZVO has antispasmodic, antimicrobial, cytotoxic and phytotoxic properties which may be due to the presence of various chemical constituents. The results strongly support the ethno medicinal use of this valuable plant in the treatment of diarrhea and various microbial infections. However more detail study is required to establish the active constituents responsible for these activities.

We are thankful to HEC (Higher Education Commission) of Pakistan for financial support for this research work.