Biological activities and Phytochemical analysis of Zanthoxylum armatum DC. leaves and bark extracts collected from Kumaun region, Uttarakhand, India

The methanolic and chloroform extracts of leaves and bark of Zanthoxylum armatum DC. were evaluated for their phytochemical analysis and biological activities. In phytochemical analysis, fargsin was identified as major constituent in leaves methanolic and leaves chloroform extracts. t-butylamine and benzoxazole, 2-(isobutylamino) were identified as the major constituents in bark methanolic and bark chloroform extracts respectively. Both the extracts exhibited moderate antioxidant activity with IC 50 values ranging from 19.42±0.07 to78.01±0.31 µg. These extracts also possess moderate anti-inflammatory activity with IB 50 values ranging from 28.53 ± 0.06 to 89.80 ± 0.05 µg. Moderate anti-bacterial activity against E. coli and S. aureus has also been observed in both the extracts. The total flavonoids, orthodihydric phenols and phenolic contents were also quantified in the extracts. Based on these observations, it can be concluded that Zanthoxylum armatum DC. may be used as herbal antioxidant, food preservative, natural anti-inflammatory drug and natural bactericidal, besides generation of data base for judicious exploitation in future.


INTRODUCTION
for industries, medicinal plants and culinary applications. Almost all the species of genus Zanthoxylum have great ability to produce tires which could be used as encapsulates in the pharmaceutical industry, diluents and emulsifying agents [4,5,6,7,8]. Zanthoxylum armatum DC., commonly called as 'Prickly ash' or 'Timur' or 'Kababe Tejal', is a shrub or small tree which predominately grows in well drained alluvial, black soil and have a strong aroma. The plants are armed scandent or erect, 6 m tall or more, with dense foliage [9]. In India, it is found in the warmer valleys of the Himalaya from Jammu and Kashmir to Assam and Khasi (1,000 to 2,100 m), in the Eastern Ghats in Orissa and Andhra Pradesh (1,200 m) and the lesser Himalayan regions in the northeastern part of India for example, Naga Hills, Meghalaya, Mizoram, and Manipur [10,11,12,13]. Essential oils and different extracts of aqueous ethanol, dichloromethane, acetone, methanol, petroleum ether have been reported to shows many biological activities viz; larvicidal, antiviral, antifungal, keratolytic, anti-protozoan, pesticidal/insecticidal, hepatoprotective, antibacterial, antihelminthic and allelopathic [14,15]. The fruits and seeds are extensively used as tonic in fever, dyspepsia and cholera, eliminate pain, used to treat heart diseases, piles, diseases of mouth, teeth and throat disorder and the bark is used for intoxicating fishes [16]. We already have reported the chemical composition and biological activities of seeds, bark and leaves essential oil of Zanthoxylum armatum DC. collected from different altitudes of Kumaun region, Uttarakhand [17,18,19]. The present study assesses the chemical composition and biological activities of methanolic and chloroform extracts of Zanthoxylum armatum DC. leaves and bark collected from Kumaun region of Uttarakhand (India).

Collection of Plant Material
The plants material was collected from Aadi Kailash region (Bhimtal) (1370 m elevation), Nainital, Uttarakhand in the month of July, 2017. The plant material was properly identified by one of the author and Plant Taxonomist (DSR) and the identity was further confirmed by comparing the specimens with authentically identified specimens at the herbarium of Botanical Survey of India, Dehradun, India (BSD). The voucher specimen (GBPUH-917/28.

Preparation of Extracts
The bark and fresh leaves of Z. armatum were finely chopped, and shade dried and grinded. The resultant powdered material (250g) was subjected for extraction by cold percolation method in organic solvents with varying polarity. The solvents from extracts were evaporated by using rotatory vaccum evaporator and the final yield of the extracts was weighed. The yields in percentage (w/w) obtained were 2.4% leaves methanol extract, 4.8% bark methanol extract, 7.2% leaves chloroform extract and 4.4% bark chloroform extract.

GC-MS Analysis
GC/MS analysis of the different extracts was performed using a GC MS-QP 2010. The GC capillary column DB-5 (30 m × 0.25 mm i.d.; 0.25 µm film thickness; J&W Scientific, Agilent, Santa Clara, CA, USA) was used. Helium was used as a carrier gas with a flow rate of 1.21 mL/min, at a pressure of 73.3 kPa. The extracts were injected at temperature: 260 °C with oven temperature programme as: Initial temperature 60°C, RAMP 3°C/min upto 210°C (isotherm for 2 min), then RAMP 6°C/min upto 280°C (isotherm for 2 min), finally hold for 11 min. The compounds were identified with the help of NIST-MS, FFNSC Wiley Library, and comparing the data with literature reports and retention indices (RI) [20].

DPPH (1, 1-diphenyl -2 -picryl -hydrazyl) radical scavenging activity
This activity was evaluated according to the developed protocols with slight modifications [21,22,23]. The tested extract samples (50-250 µg/mL) were taken and mixed with 5 mL of a 0.004% methanolic solution of freshly prepared DPPH. The O.D. (optical density/optical absorbance) was measured by using UV-visible spectrophotometer (Thermo Scientific EVOLUTION-201 series) at 517 nm. All the observations were recorded in triplicate with reference to the standard antioxidants catechin and BHT. Inhibition of free radical in percent (IC%) was calculated by using the equation: IC% = (A 0 -A t )/A 0 × 100 where, A 0 = absorbance value of control sample, A t = absorbance value of test sample, IC = inhibitory concentration. Percent inhibition was plotted against concentrations in graph. The standard curve was drawn using standard antioxidant (BHT and catechin) to calculate the IC 50 values for different extracts and standard.

Reducing power
The reducing power of extracts was evaluated by the method developed earlier and is being practiced [24]. Various amount of extracts (50-250 µg/mL) were mixed with 2.5 mL of phosphate buffer (pH= 6.6, 200 mM,) and 2.5 mL of 1% potassium ferricyanide, K 3 [FeCN 6 ]. After 20 minute incubation at 50±1ºC, 2.5 mL of trichloroacetic acid was added to the mixtures, followed by centrifugation at 650 rpm for 10 min. The supernatant (1 mL) was mixed with 5mL distilled water followed by 1 mL of 0.1% ferric chloride. The absorbance of the resultant solution was measured spectrophotometrically at 700 nm. All the readings were recorded in triplicate. Ascorbic acid was used as standard. The percent reducing power of samples was calculated using the formula: Reducing power % = (A 0 -A t )/A 0 × 100 where, A 0 = absorbance value of control sample, A t = absorbance value of test sample. Percent inhibition was plotted against concentrations in the graph. The standard curve was drawn using standard antioxidant (BHT) to evaluate the RP 50 values for standard and different extracts.

Metal chelating activity
The chelation of Fe+2 was evaluated using the method developed earlier [24]. 0.1 mL of 2mM FeCl2.4H2O, 0.2mL of 5mM ferrozine and 4.7 mL of methanol was added to various concentrations of test sample (50-250 µg/mL). The solutions were mixed thoroughly and incubated for 10 min. At 562 nm, the absorbance of test sample was measured in a UV spectrophotometer (Thermo Scientific EVOLUTION 201 series). All the readings were recorded in triplicate; EDTA (0.01 mM) was used as the standard. The metal-chelating activity of tested samples, expressed as percentage was calculated by using the formula: IC% = (A 0 -A t )/A 0 × 100 where, A 0 = absorbance value of control sample, A t = absorbance value of test sample, IC = inhibitory concentration. The percent of chelating ability was plotted against concentrations in graph. The standard curve was drawn using standard antioxidant (EDTA) to calculate the IC 50 values for standard and different extracts.

Estimation of Phenols
The phenolic assay of both the extracts of Z. armatum was calculated quantitatively by spectrophotometer in terms of total phenols, flavonoids, and orthodihydroxy phenols. The concentrations of these samples were measured with the help of working calibration curve by the relation among concentration and absorbance of the sample.

Total phenolic assay
The total phenols were determined by the Folin-Ciocalteu reagent (FCR) method [25]. In brief, 1 mL of the sample extract was poured into a test tube and mixed with 1 mL of 80% methanol and 8 mL of distilled water. In each sample 0.5 mL of 1 N FCR was added and mixed. After 5 min., 1 mL of saturated Na 2 CO 3 was added to the reaction mixture and allowed to stand for 60 min. The absorbance of test sample was observed at 650 nm. The standard curve was drawn using various concentrations of gallic acid and results were expressed as mg of gallic acid per gram of sample in dried weight.

Estimation of flavanols
Aluminum chloride colorimetric assay [26] was applied for the estimation of flavanols. 10 mg of extract were dissolved in 10 mL of 80% methanol to prepare stock solution. In a test tube, 0.1 mL of stock solution was mixed with 1.25 mL water and 0.75 mL of 5% NaNO 2. The mixture was incubated for 5 min. 0.15 mL of 10% AlCl 3 was added to the mixtures after incubation. After 6 min, 275 µL of distilled water and 0.5 mL of 1 N NaOH were added, after thoroughly mixing of the solution. At 510 nm, the intensity of pink colour was obtained. The standard curve was established using different concentrations of catechin and the concentrations were evaluated with the help of calibration curve and expressed in mg/100gm of dry material [27].

Estimation of orthodihydric phenols (OHP)
10 mg of extract was added in 10 mL of 80% methanol to prepare stock solution. 0.1 mL of the extract solution was poured in a test tube and mixed with 0.4 mL of water and 1 mL of 0.05N HCl, 1 mL of Arnow's reagent (10 g sodium nitrite and 10 g sodium molybdate made up to 100 ml with distilled water), 10 mL of water and 2 mL of 1 N NaOH. The resultant solutions were thoroughly mixed (appearance of pink colour) and at 515 nm absorbance was measured. The standard curve was prepared with the help of working standard catechol solution at different concentrations. The concentration was calculated and expressed in mg per 100gm of material [28].

In-vitro Anti-inflammatory Activity
The in-vitro anti-inflammatory activity of extracts was evaluated by using inhibition of albumin denaturation technique, by the standard protocols [29,30,31]. The reaction mixture (5 mL) was comprised of 0.2 mL of egg albumin, 2.8 mL of phosphate buffer solution (pH= 6.4) and 2 mL of varying amount of extracts (25 µg/mL, 50 µg/mL, 75 µg/mL, 100 µg/mL, 125 µg/mL, 250 µg/mL and 500 µg/mL). Double distilled water was used as control. At 37±2°C, the mixtures were incubated in a BOD incubator (for 15 min and then heated for 5 min at 70°C in water bath). Subsequent to cooling, the absorbance was observed at 660nm. All the readings were observed in triplicate, diclofenac sodium was used as the standard. The percentage inhibition of protein denaturation was calculated using the formula: IB% = (A 0 -A t )/A 0 × 100 where, A 0 = absorbance value of control sample, A t = absorbance value of test sample, IB = inhibitory concentration. The extract/drug concentration for 50% inhibition (IB 50 ) was determined by plotting percentage inhibition with respect to control against treatment concentration.

Antibacterial Activity
The antibacterial activity was determined by using Agar well diffusion method [32,33]. It was expressed as the mean of zone of inhibition (ZOI) diameters (mm) produced by the various extracts. For screening plates were prepared by using nutrient agar. The inoculums (50µL) of different bacterial strains were spread evenly on respective plates with sterile spreader. A borer (8mm diameter) was used to cut well. 20µL of different concentrations of the extracts were poured in each well and incubated at 24 hrs at 37±2°C. The diameter of ZOI was measured and the mean was recorded. The experiment was performed in triplicate.

Statistical Analysis
The data were analyzed by using Analysis of Variance (ANOVA) using STPR. All the values were taken in triplicate. IC 50 was determined by linear regression analysis using MS Excel 2007.
The literature search reveals no report on GC/MS analysis of chloroform and methanolic extracts of Z. armatum leaves and bark (ZNLClE, ZNLME ZNBME and ZNBClE) hence is being reported first time. The major fargsin detected in leaves has also been reported in the leaves essential oil of Z. acanthopodium from north-eastern region of India [34]. Hence, the present analysis reveals the first report on it. The study on comparative chemical composition among ZNLME, ZNBME, ZNLClE and ZNBClE has been represented in Table 1 and structures of major chemical constituents in extracts of Z. armatum has been illustrated in Figure 1.

Total phenols
The total phenolics content in different extracts of Zanthoxylum armatum DC. were observed in the order of: ZNLME (31.23±0.03 mg/g GAE) > ZNBME (27.58±0.05 mg/g GAE) > ZNBClE (25.02±0.04 mg/g GAE) > ZNLClE (17.74±0.02 mg/g GAE). The variation in total phenolic content in different extracts might be because of different solubility of phenolics in organic solvents with varying polarity. Among different plant extracts from different parts of plant, it has been observed that ZNLME contained maximum total phenolic content (31.23±0.03 mg/g GAE) while ZNLClE showed minimum among the extracts (17.74±0.02 mg/g GAE) ( Table 2).

Total flavonoids
Flavonoids are one of the most important groups of bioactive secondary metabolites in plants and are known for their health promoting propertied due to protective effects against cancer, cardiovascular disease and other diseases [35]. In the present study, the total flavonoids content in different extracts were observed in the order of: ZNLClE (77.18±0.06 mg/g CNE) > ZNBME (68.04±0.06 mg/g CNE) > ZNBClE (56.72±0.03 mg/g CNE) > ZNLME (47.63±0.14 mg/g CNE) ( Table 2).
The total phenolics and flavonoids in bark ethanolic extracts from Pakistan have also been reported [36]. The results revealed the presence of total phenolics (11.66±0.33mg/g) and flavonoids (13.68±0.66mg/g) in leaves and total phenolics (16.48±1.33mg/g) and flavonoids (18.33±1.22mg/g) in bark.
Our study revealed high phenolic and flavonoids content in leaves methanolic extract (31.23±0.03 mg/g GAE and 47.63±0.14 mg/g GAE respectively) and bark methanolic extract (27.58±0.05 mg/g GAE and 68.04±0.06 mg/g GAE respectively) compared to Barkatullah et al., 2017 [36] (Table 2). This discrepancy in results might be due to diverse natural habitats of sampled plants.

DPPH radical scavenging activity
All the extracts exhibited good radical scavenging activity as a function of their concentrations. ZNLME ( (Table 3) Kanwal et al., 2015 [37] has reported the moderate antioxidant activity of Z. armatum leaves methanolic extract by DPPH and reducing power assay. Present study exhibited good antioxidant assay in all the extracts. Among all the extracts ZNLME exhibited maximum antioxidant activity while the minimum was found in ZNLClE. The phenolics are found to be related and reported to possess the antioxidant activity [37]. In present study, a good quantitative amount of phenolics, flavonoids and orthodihydric phenols have been observed. Hence, the good antioxidant activity of extract is because of the presence of phenols. Further, it has been observed that the phenolic content in the extract exhibited positive correlation with antioxidant activity and negative with IC 50 values. The antioxidants are used to prevent the oxidative deterioration of foods and food products and can be used as food preservation for long-life of the food material. The synthetic antioxidants possess deleterious effects. Hence, in present scenario, the herbal antioxidants are in demands. Based on these facts, it can be concluded that the medicinal herb Z. armatum can be used as an herbal source of natural antioxidants and phytochemical source of nutraceuticals.

In-vitro Anti-inflammatory Activity
The in-vitro anti-inflammatory activity of extracts was performed by inhibition of egg albumin denaturation method as described in materials and methods section. Denaturation of proteins is well documented and is caused by inflammation process, mostly in conditions like arthritis. In protein denaturation mechanism, due to external stress, influence of chemical reactions results in distortion of protein's tertiary and secondary structure and leads to denaturation of proteins [38].
As the part of study on mechanism of anti-inflammation activity, capability of plant extracts were studied. It was observed that the extracts ZNLClE, ZNLME, ZNBClE and ZNBME inhibited the heat induced albumin denaturation in a dose dependent manner as monitored spectrophotometrically.
The in-vivo anti-inflammatory activity of ethyl acetate extract from stems and roots of Z. armatum has been reported. The study revealed that the constituents like eudesmin, pinoresinol, sesamin and yangambin were responsible for in-vivo anti-inflammatory activity [39]. In present study most of the extracts exhibit moderate anti-inflammatory activity. The compounds eudesmin, sesamin and yagambin are also present in our extracts. Hence, it can be inferred that the anti-inflammatory activity is possibly due to the presence of these compounds or synergetic effect of co-occurrence of major, minor or trace constituents. Presence of polyphenolic compounds such as alkaloids, flavonoids, tannins, steroids, and phenols has also been reported to posses anti-inflammatory  activity [40]. It has been reported that the total phenolics and antioxidants possess anti-inflammatory activity [41,42]. There is a positive correlation between antioxidant activity and total phenolics in present study. Hence, the constituents responsible for antioxidant activity may also be responsible for anti-inflammatory activity, i.e. there is positive co-relation between anti-inflammatory activity and phenolics/antioxidants.

Antibacterial Activity
The antibacterial efficiency of the ZNLClE, ZNLME, ZNBClE and ZNBME has been presented in Table 5. The extracts were tested for antibacterial activity against gram-positive and gramnegative bacteria and were found to be effective against all the tested bacterial strains as compared to the antibiotic gentamicin sulphate, taken as standard. It was observed that ZNLME showed maximum ZOI (zone of inhibition) of 17.67 mm at 500 ppm against E. coli while, ZNBME exhibits maximum ZOI of 17.67 mm at 500 ppm against S. aureus. The minimum ZOI was observed in ZNBClE (5.33 mm), at 250 ppm against E. coli while, in case of S. aureus ZNBClE showed minimum ZOI (3.67 mm) ( Table 5). The screening of antibacterial activity of different extracts of Z. armatum against E. coli has been represented in Figure 3 while the screening of antibacterial activity of different extracts of Z. armatum against S. aurens has been represented in Figure 4.    [44] reported the antibacterial activity against Staphylococcus aureus, Escherichia coli, Proteus vulgaris and Pseudomonas aeruginosa in Z. armatum chloroform, methanol and acetone bark extracts collected from lesser and higher Himalaya (altitude 700-2000 m). The acetone and methanol extracts of bark were found to be more effective for S. aureus and chloroform extract for P. vulgaris.

CONCLUSIONS
The above results show that the Z. armatum is a good source of major compounds like eudesmin, sesamin, methyl-vanillin, linolenic acid, fargsin, γ-sitosterol, doxepin, besides other major and minor constituents. These compounds find their wide applications in perfumery, preservation, pharmacological activities and starting material for the synthesis of novel molecules. The compounds like doxepin and (Z, Z) -6, 9-cis-3, 4-epoxy-nonadecadiene has also been reported to possess anti-depressant activity and sex pheromone, respectively. The extracts have also been found to possess moderate antioxidant, anti-inflammatory and antibacterial activity. The total flavonoids, orthodihydric phenols and phenolic contents were also quantified in methanolic and chloroform extract of leaves and bark. In present scenario, food and pharmaceutical industries are in search of environmentally benign novel lead molecules from herbal origin. The present study concludes that the entire plant of Zanthoxylum armatum might be used as a good source of herbal antioxidants, food preservative, natural anti-inflammatory drug and natural anti-bacterial agent after proper clinical trials. The present study contributes for preparation of database on this species so that it can be exploited judiciously and scientifically.