PHYTOCHEMICAL SCREENING, CHARACTERIZATION OF ESSENTIAL OIL AND ANTIMICROBIAL ACTIVITY OF SCHINUS MOLLE (ANACARDIACEAE) COLLECTED FROM EASTERN HARARGHE, ETHIOPIA

1Department of Food Science and Postharvest Technology, Institute of Technology, Haramaya University, Ethiopia, P. O. Box 138, Dire Dawa. 2School of Plant Sciences, College of Agriculture and Environmental Sciences, Haramaya University, Ethiopia, P. O. Box 138, Dire Dawa. 3Department of Chemistry, College of Natural and Computational Sciences, Haramaya University, Ethiopia, P. O. Box 138, Dire Dawa. Authors: Tesfahun Lamboro1, Melese Mengistu2 and Teshome Gonfa Hordofa3 PHYTOCHEMICAL SCREENING, CHARACTERIZATION OF ESSENTIAL OIL AND ANTIMICROBIAL ACTIVITY OF SCHINUS MOLLE (ANACARDIACEAE) COLLECTED FROM EASTERN HARARGHE, ETHIOPIA Original article


Introduction
For thousands of years, human beings used plants to treat various ailments and still many peoples from developing countries rely on traditional doctors and the collections of medicinal plants to treat health problems (Mehani and Segni, 2013). The medicinal plants are the plants whose parts (leaf, seed, fruits, stems, roots, barks), extracts, infusions, decoctions, powders are used in the treatment of different diseases of humans, plants and animals (Samy, 2005). The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human or animal body part of the plant. Extracts of many plants are highly efficient against parasitic as well as microbial infections (Bharti et al., 2013;Jameela et al, 2016;Parekh and Chanda, 2006). Unlike synthetic pesticides and fungicides, antimicrobials of plant origin are not associated with many side effects and are environmental friendly.
The essential oils are volatile aromatic substance extracted from the plant. They are produced in aromatic and medicinal plants as secondary metabolites and are obtained from various plant parts (flower, seed, leaves, bark, herbs, wood, fruits and roots) and stored in secretory cells, cavities, vessels, or epidermal cells called glandular trichomes (Pedro, 2012). They have many therapeutic properties. In herbal medicine, they are used for their antiseptic properties against infectious diseases of fungal and bacterial origin (Mehani and Segni, 2013). The inherent activity of an oil extract may be related to its chemical composition, the proportions of the components and the interactions between them. The chemical composition of the essential oil consists mainly of monoterpene hydrocarbons (e.g., α-pinene, β-pinene, sabinene, terpinen-4-o), and some sesquiterpenes such as (+) spathulenol and germacrene-D (Lisin et al., 1999). Essential oils act against microorganisms often causing instability in the plasma membrane leading to cell lysis. Although the antimicrobial activity can be triggered by a single chemical compound, it usually appears to result from a synergy between several chemicals in the oil (Bhavanani and Balow, 1992;Elhayouni et al., 2008).
Schinus molle L. (Anacardiaceae) is one of very important medicinal plant native to Argentina, Bolivia and Peru but has since been introduced to Ethiopia and known with local name as "Kundo Berbere" (Amharic) (Basil and Zakaria, 2014). This plant plays an important role in pharmacology and pharmaceutical chemistry because of its high essential oil content in its different parts (Belhamel et al, 2008). In Ethiopia, especially in Eastern part of Ethiopia, the fresh leave was traditionally used to repel insects. The insecticide effect of this plant might be combination of bio active intergradients produce a physiologic action on the insects.
Some studies have concluded that the Essential oils as a whole have much greater antibacterial activity than a mixture of the major components, which suggests that minor components are critical for this activity and may have a synergistic effect or potentiating influence (Burt, 2004;Ljalem and Unnithan, 2014;Marongiu et al, 2004). However, in Ethiopia, there is no sufficient work on antimicrobial effect of leaf, stem bark, fruit and root bark extract of S. molle on growth and activity of different fungal and bacterial strains. Therefore, this study was aimed to screen phytochemicals, investigated the chemical constituent of essential oils and evaluates antimicrobial activities of leaf; stem and root bark extracts of S. molle against selected plant pathogens (Fusariumverticillioides, Aspergillusniger, Xanthomonascampestris, pv. Campestris and Ralstoniasolanacearum).

Plant Material Collection
The aerial parts (stem bark and leaf) and root bark of Schinus molle plant were collected from Eastern Hararghe, Ethiopia. Fresh plant materials were thoroughly washed under tap water, dried with blotting paper and made ready for extraction. The plant species deposited at the National Herbarium of the Addis Ababa University, Ethiopia and a voucher specimen (No. TT-12) has been given.

Preparation of Plant Extracts
Fresh plant parts (stem and root barks and leaf of Schinus molle) were collected and thoroughly washed under tap water and shade-dried at room temperature or three weeks. The dried plant parts were crushed to fine powder using an electric grinder. Exposure to direct sunlight was avoided to prevent the loss of active components. The powder of collected plant materials (50 grams of each plant parts) were extracted successively with 600 mL of n-hexane, chloroform and ethanol for 6hours using Soxhlet extraction. Each solvent was removed by rotary flash evaporator at lower temperature under reduced pressure and the crude extracts were stored in dark vials at 4°C for future uses.

Hydro-distillation and purification of essential oil
The fresh leaf and stem bark of Schinusmolle (600mg) was soaked in water at a ratio of 1:2 (w/v) and subjected to hydro-distillationfor 6hours using a modified Clevenger type glass apparatus. The essential oil was separated from the aqueous layer by separatory funnel, using the non-polar chloroform as a solvent. The residual oil was dried over anhydrous Na 2 SO 4 and kept in a refrigerator (5°C) for subsequent experiments. Analysis of essential oil was carried out by Clarus 600/600 P Gas Chromatograph/Mass Spectrometer (Perkin Elmer, USA) system.

Microbial Culture preparation
Bacterial species (Xanthomonascampestris, pv. Campestris and Ralstoniasolanacearum.) were used for the antibacterial screening in this study. These organisms were maintained on agar slope at 4 o C and sub-cultured for 24hr before use. Similarly, the fungal plant pathogens Fusarium verticillioides and Aspergillus niger were used for the antimicrobial test of the extract.

Microbial Susceptibility Testing
Standardized inoculums with 0.5 McFarland standards were introduced onto the surface of sterile agar plates, and a sterile glass spreader was used for even distribution of inoculums. McFarland is a barium sulphate standard against which the turbidity of the test and controlled inoculum was compared. McFarland was prepared by mixing two, solution "A" is 1 % v/v solution of sulphuric acid (H 2 SO 4 ) and solution "B" is 1 % w/v solution of barium chloride (BaCl 2 ). To get 0.5 McFarland standard, concentration equivalents to cell density of about 10 7 -10 8 CFUg -1 , the amount of 0.5 ml BaCl 2 of 1 % solution "A" was mixed with 99.5 ml H 2 SO 4 of 1 % solution "B" (Bauer et al., 1966). A sterile paper disc previously soaked in known concentration of extracts will be carefully placed at the centre of the seeded labeled agar. The plates were incubated aerobically at 37 o C and examined for zone of inhibition after 24 hrs. Each zone of inhibition was measured with a ruler and compared with the control (Bauer et al., 1966).

Determination of Total Tannin and Phenol Content
Tannin Condensed tannin was analyzed by vanillin-HCl method of (Price et al., 1980) using the modified Vanillin-HCl methanol method. The Vanillin-HCl reagent was prepared by mixing equal volume of 8% concentrated HCl in methanol and 1% Vanillin in methanol. The solutions of the reagent were mixed just prior to use. About 0.2 g of the ground sample was placed in small conical flask. Then 10 mL of 1% concentrated HCl in methanol was added. The conical flask was capped and continuously shaken for 20 min and the content then is centrifuged at 2500 rpm for 5 minutes. About 1mL of the supernatant was pipette into a test tube containing 5 mL of Vanillin-HCl reagent. Absorbance at 450 nm was read on spectrophotometer after 20 minutes incubation at 30 0 C. A blank sample was also analyzed and its absorbance was subtracted from sample absorbance. A standard curve also prepared from catechin (1 mg/mL). Tannins content was expressed as catechin equivalent as follows: Where: C is concentration corresponding to the optical density, 10 is volume of the extract (mL), 200 is sample weight (mg).

Phenolic
Total phenolic content was determined according to Folin-Ciocalteu method (Sharma and Gupta, 2010). Sample 200 mg was extracted with 4mL of acidified methanol (HCL/Methanol/water, 1:80:10 v/v/v) at room temperature (25 O C) for 2 hrs. Aliquot of extract (200 µL) was added to 1.5 mL freshly diluted (10 fold) Folin-Ciocalteu reagent. The mixture was allowed to equilibrate for 5 min and then mixed with 1.5 mL of sodium carbonate solution (60 g/l). After incubation at room temperature (25 O C) for 90 min, the absorbance of mixture was read at 725 nm (6505 uv/vis spectrophotometer, Model 6505, U.K, GENWAY). Acidified methanol was used as a blank. Form the stock solution of standard gallic acid, a series of six standard solutions (0, 5, 10, 15, 20, 25, 30 and 35 ppm) was prepared. The amount of total phenolic was estimated from the calibrated curve as gallic acid equivalent (GAE) in milligram per kilogram of sample. Total phenolic content was calculated by the following formula: Where: µg/mL is the absorbance reading concentration, Df is dilution factor.

Procedure of Phytochemical Tests
The different qualitative chemical tests were performed for establishing the profile of given extracts to detect various phyto-constituents present in them. The phytochemical were analyzed according to standard screening tests using conventional procedures.

Test for Flavonoids
Lead acetate Test: -Extracts were treated with few drops of lead acetate solution. Formation of yellow colour precipitate indicates the presence of flavonoids.

Test for Alkaloids:
Wagner's Test: -Filtrates were treated with Wagner's reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicated the presence of alkaloids.
Hager's Test: -Filtrates were treated with Hager's reagent (saturated picric acid solution). Presence of alkaloids was confirmed by the formation of yellow coloured precipitate.

Detection of Saponins
Foam Test: 0.5 gm of extract was shaken with 2 mL of water. If foam produced persists for ten minutes it indicates the presence of saponins.

Test for Steroid
5 drops of concentrated H 2 SO 4 was added to 1cm 3 of the extract. A reddish brown colour indicates the presence of steroids.

Test for Terpenoids
Salkowski test; -5 mL of various solvent extract was mixed in 2 mL of chloroform followed by the careful addition of 3 mL concentrated (H 2 SO 4 ). A layer of the reddish brown coloration was formed at the interface thus indicating a positive result for the presence of terpenoids.

Detection of Phenols
Ferric Chloride Test: Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenols.

Test for Quinones
One ml of each of the various extracts was treated separately with ferric chloride solution. Quinines give coloration ranging from red to blue.

Test for Carbohydrates
Molisch test: Treat extract with few drops of alcoholic alpha-naphthol. Add 0.2mL concentrated sulphuric acid slowly along the sides of test tube, purple to violet colour ring appears at junction was showed the presence of carbohydrates.

Detection of proteins and amino acids
Xanthoproteic Test: -The extracts were treated with few drops of conc. nitric acid. Formation of yellow colour indicates the presence of proteins.
Ninhydrin Test: -To the extract, 0.25% w/v ninhydrin reagent was added and boiled for few minutes. Formation of blue colour indicates the presence of amino acid.

Data Analysis
The data was analyzed by using simple statistical methods. Experimental results were expressed as means ± SD. All measurements were replicated three times.

Phytochemical screening of the crude extracts
The different qualitative chemical tests were performed for screening the profile of given extracts to detect various phyto-constituents present in the plant. The phytochemicals were analyzed according to standard screening tests using conventional procedures. The results of constituents of the crude extract of parts of Schinus molle were presented in table 1. Phytochemical screenings results (Table 1) of crude extracts of leaf of Schinus molle revealed moderate presence of flavonoids, alkaloids, steroids, phenols and carbohydrates. The crude extracts of steam bark showed strong presence of quinones and carbohydrates and moderate presence of flavonoids, alkaloids, steroid and phenols. Phytochemical screenings of crude extracts of root of schinus molle also showed strong presence of terpenoides, quinones and carbohydrates and moderate presence of flavonoids. Flavonoids were present in both ethanolic and chloroform extracts of leaf, stem and root barks of Schinus molle. Similarly, alkaloids were found in both chloroform and ethanol extracts of leaf and stem barks of Schinus molle. They were also found in chloroform extracts of root barks while absent in ethanol extracts. Terpenoids were found in petroleum ether extracts of all plants parts while steroids were found in both petroleum and chloroform extracts of leaf and stem barks. Quinones were also present strongly in ethanolic extract of the above-mentioned plant parts. On the other hand, carbohydrates were found all petroleum and chloroform extracts of only all plant parts.

Tannin and Phenolic content of the different parts of Schinus molle
The result of experiment for total tannin and phenol contents from the leaf, stem and root bark was indicated in table 2. This result showed that, the methanolic extracts of the leaf, root and stem bark of Schinus molle showed high amount of total phenolic content when compared to the total tannin in the three parts of the plant. Similar finding was reported by Kasmi et al, (2016) there by higher phenolic content was observed on the fruit of Schinus molle with aqueous solution extracts.
Our findings showed that Schinus molle is rich in phenolic compounds which were mainly responsible for several antimicrobial activities due to the trapping potential of free radicals and activation of antioxidants of the cells. Phenols activate the natural defensive mechanism on the microbes.

Antimicrobial activity of leaf, stem and root bark extract of Schinus molle
The result for antimicrobial effects of extracts from leaf, stem and root bark of Schinus molle against bacterial and fungal plant pathogens were summarized below (Table 3 and 4). The result revealed that stem and root bark ethanol extracts exhibited relatively higher (11.3mm) inhibition zone against Xanthomonas campestris, pv. Campestris. The n-hexane extraction of leaf and stem showed relatively less inhibition against Xanthomonas as compared to others. Ralstonia showed relatively less inhibition zone (1.7 and 2.7mm) against CF extracts of stem and root. The CF leaf extracts and the n-hexane root extracts showed relatively higher inhibition against Ralstonia. As compared to Xanthomonas, less inhibition of the plant extracts against Ralstonia were observed (Table 3). Relatively higher zone of inhibition (10mm) was observed by the ethanolic extracts of the root of Schinus molle against Fusarium verticillioides followed by the chloroform extracts of the root (8mm) against Aspergillus niger. The least zone of inhibition was detected in the chloroform extracts of the root, stem and leaf extracts against Fusarium verticillioides. The n-hexane and chloroform extracts of the plant showed relatively higher zone of inhibition against Aspergillus niger than Fusarium verticillioides (Table 4).
When the overall zone of inhibition was examined, the crude extracts showed relatively higher zone of inhibition against the tested bacterial species than fungal species. The ethanol extracts of stem and root showed relatively higher zone of inhibition against the tested bacteria than the other solvent extracts. Overall, the maximum inhibition zone of crude extract in the experiment was (12mm).
A report from Rhouma, et al, (2009) in the antimicrobial activity of leaf extracts of Schinus molle against Pseudomonas savastanoi pv. Savastanoi showed similar results and the zone of inhibition were between 9-12mm. Another report from (Mehani and Segni, 2013) on the antimicrobial activity of plant extract of Schinus molle on four bacterial pathogens (E. coli, Pseudomonas aeruginosa, Staphyloccus and Klebsiella pneumonia) with a zone of inhibition 13, 12.33, 11.67 and 10.5mm, respectively which is in agreement with the current finding.
This result could be due to the polarity of the compounds which were extracted by each solvent and the ability of extracts to diffuse and dissolve in different culture media used in the study. Absence of antimicrobial activity does not mean that the bioactive compounds are not present in the plant or the plant has no activity against microorganisms. Presence of inadequate quantities of active constituent or constituents in the extract to exhibit the antimicrobial activity can be the reason for the negative results.

Conclusion
Phytochemical screening results of crude extracts of stem and root bark of Schinus molle revealed strong presence of quinones and carbohydrates and moderate presence of flavonoids, alkaloids, steroids and phenols. As compared to chloroform and n-hexane, stem and root bark ethanol extracts exhibited relatively higher (11.3mm) inhibition zone against Xanthomonas campestris, pv. Campestris. Relatively higher zone of inhibition (10mm) was observed by the ethanolic extracts of the root of Schinus molle against Fusarium verticillioides followed by the chloroform extracts of the root (8mm) against Aspergillus niger. Furthermore, the leaf extract of the plant were characterized and showed three alcohols, three sesquiterpene hydrocarbons, one aromatic hydrocarbon and three oxygenated sesquterpenes, totally 91.78%) % of the essential oil were identified by GC-MS. The GC-MS analysis showed, three terpenoids namely monoterpene (66.02%) as a major components, sesquiterpene hydrocarbons (13.63%) and oxygenated sesquterpenes (11.07%) were identified from the stem bark. The finding indicated that essential oils from different parts of Schinus molle have a promising potential on inhibiting activity of pathogenic microbes.