Phytochemical screening and antibacterial activity of Tulipa Systola stapf. collected in Kurdistan region-Iraq

Article History: Received: 07/07/2015 Accepted: 19/04/2016 Published:10/10/2016 Tulipa systola Stapf. is a monocotyledonous plant in the Liliaceae family, collected in Kurdistan Region-Iraq which is uses by Local people as a pain killer and wound healing. In this study; antibacterial activity and phytochemical qualitative screening of ethanolic extracted from roots, leaves and flowers of Tulip were studied. The results revealed that the ethanol extracts of Tulipa Systola exhibited strong antibacterial activity against both gram positive and gram negative bacteria’s.


INTRODUCTION
The Tulip is a monocotyledonous plant in the Liliaceae family.The number of species ranges from about 45 (stork 1984) to more than 10 (Hall 1940).According to the taxonomic classification by (Van Raamsdonk 1995), the genus is divided into two subgenera: Tulipa and Eriostemones (Anderson 2006).The genus Tulipa (Liliaceae) is of great economic, horticultural, esthetical, ecological, conservational, and taxonomic interests.Tulips are present and distributed from the southern Balkans to Siberia and west China, North Africa (Algeria), the east Mediterranean, East Iran, Israel, Jordan and Turkey (Veldkamp 2012).In this study the ethanol extracts of roots, leaves and flowers of Tulipa Systola Stapf.were studied for some secondary metabolites like, saponins, tannins, alkaloids, anthraquinones, flavanoids, glycosides and cardiac glycosides according to the methods described by (Mohammed 2009) and (Chaudhari 2006).Each of the test result was presented as negative (−) or positive (+).On the other hand the antibacterial activities of the ethanol extracts were investigated.

MATERIALS AND METHODS: PLANT MATERIALS:
Tulipa systola Stapf.was collected in April 2014 from Korek Mountain near to Rewanduz district -Erbil / Kurdistan region from northern Iraq.The materials were identified and classified by botanists.A voucher specimen (7201) was deposited at Education Salahaddin University Herbarium (ESUH), Kurdistan.The plant raw materials (Roots, Leaves and Flowers) were washed and air-dried under shade at room temperature (20-25 o C).After drying, the plant parts were grounded into fine powder using a laboratory grinding mill, to provide homogeneous powder for analyzing.

A R T I C L E I N F O A B S T R A C T
Powdered materials were stored in bottles in a dark room temperature and then used.

EXTRACTION WITH ETHANOL:
The defatted roots, leaves and flowers part residue were extracted with (500ml) of ethanol using ultrasound for (30min) then macerated for (3hrs) with continuous stirring at room temperature.The procedure repeated three times for each part separately.Then the mixtures were filtered and the solvent removed under "vacuum" using rotary evaporator affording a crude ethanol extracts (TR1 root, TL1 leaves and TF1 flowers crude extracts).

PHYTOCHEMICAL TESTS
The phytochemical qualitative tests were performed according to the procedures described by (Mohammed 2009) and (Chaudhari 2006).

Test for Reducing Sugars (Fehling's test)
The ethanol extract (0.5 g in 5 mL of water) was added to boiling Fehling's solution (A and B) in a test tube.The solution was observed for a color reaction (a purple ring at the junction of two liquids) (Mohammed 2009).

Test for Anthraquinones
The extract (0.5g) was boiled with 10 mL of sulphuric acid (H 2 SO 4 ) and filtered while it was hot.The filtrate was shaken in 5 mL of chloroform.The chloroform layer was pipetted into another test tube, and 1 mL of diluted ammonia was added.The resulting solution was observed for color changes (Tiwari 2011).

Test for Terpenoids (Salkowski's test)
The chloroform (2 mL) was added to 0.5 g of the extract.Concentrated H 2 SO 4 (3 mL) was carefully added to form a layer, and the solution was observed for a reddish brown discoloration at the interface, which indicated the presence of terpenoids (Chaudhari 2006).

Test for Flavonoids
Four methods were used to test for flavonoids.(a) Diluted ammonia (5 mL) was added to a portion of an aqueous filtrate of the extract.Concentrated sulphuric acid (1 mL) was then added.A yellow discoloration that on standing indicated the presence of flavonoids.(b) A few drops of 1% aluminum solution were added to a portion of the filtrate.A yellow coloration indicated the presence of flavonoids (Tripathi 2011).(c) A portion of the extract was heated with 10 mL of ethyl acetate over a steam bath for 3 min.The mixture was filtered, and 4 mL of the filtrate was shaken with 1 mL of dilute ammonia solution.A yellow coloration indicated the presence of flavonoids.(d) A small piece of magnesium ribbon was added to the alcohol solution of the extract followed by drop wise addition of concentrated hydrochloric acid.The color changing from red-crimson indicates flavonols, crimson to magenta indicates flavonones and green blue indicates the test is positive (Chaudhari 2006).

Test for Saponins
Distilled water (5 mL) was added to 0.5 g of the extract in a test tube.The solution was shaken vigorously and observed for a stable persistent froth.The froth was mixed with three drops of olive oil and shaken vigorously, after which it was observed for the formation of an emulsion (Chaudhari 2006).

Test for Phenolic Compounds
The extracts (50 mg) were dissolved in distilled water and to this; 3 mL of 10% lead acetate solution was added.Formation of a bulky white precipitate indicated the presence of phenolic compounds (lead acetate test).50 mg of extract dissolved in 5 mL of distilled water and to this; 2 mL of a 1% solution of gelatin containing 10% sodium chloride was added.The formation of white precipitates indicated the presence of phenolic compounds (gelatin test) (Chaudhari 2006).TEST FOR TANNINS About 0.5 g of the extract was boiled in 10 mL of water in a test tube and then filtered.A few drops of 0.1% ferric chloride were added, and the solution was observed for brownish green or a blue-black discoloration (Chaudhari 2006).

TEST FOR ALKALOIDS
The extracts (0.5 g) were diluted with 10 mL of alcohol, acidified by hydrochloric acid then boiled and filtered.2 mL of dilute ammonia was added to 5 mL of the filtrate, followed by the addition of 5 mL of chloroform.The mixture was shaken gently to extract the alkaloid base, and the chloroform layer was extracted with 10 mL of acetic acid.The chloroform layer was divided into two portions.Mayer's reagent was added to one portion and Draggendorff's reagent to the other.The formation of a cream (with Mayer's reagent) or a reddish brown precipitate (with Draggendorff's reagent) was regarded as positive for the presence of alkaloids (Devika 2012).

Test for Cardiac Glycosides (Keller-Killiani test)
The extracts (0.5g) were diluted to 5 mL in water, and 2 mL of glacial acetic acid containing one drop of ferric chloride solution was added to it.1 mL of concentrated sulphuric acid was added to form a layer, and the color at the interphase was recorded.A brown ring at the interface indicated the presence of a deoxysugar characteristic of cardenolides.A violet ring may appear below the brown ring, while in the acetic acid layer; a greenish ring may form just above the brown ring and gradually spread throughout this layer (Devika 2012).

Total polyphenol content (Folin-Ciocalteau assay):
The The reaction mixture consisted of a solution of the reference standard or extract in 10% EtOH (1mL), deionized H 2 O (6 mL), and the Folin-Ciocalteau reagent (500 μL).The mixture was allowed to react for 3 min under stirring; then 1.5 ml of Na 2 CO 3 (20% w / v) was added, and the mixture was diluted to totally 10 mL with deionized water.The flask was then sealed and stored in the dark for two hours at room temperature.
The extracts of the plants were diluted as necessary to ensure that the final concentrations fill within the range of the calibration curve of the standard.Absorbance values were determined at 760 nm, against a blank containing 10% EtOH (1 mL).The equation of the calibration curve was (y = 0.0785x + 0.0253) and the analyses were performed in duplicate.The total polyphenol content in each sample was calculated using the formula: C = c V/m; where, C = total polyphenol content of mg gallic acid equivalent (GAE)/g dry extract, c = concentration of gallic acid obtained from calibration curve in mg/mL, V = volume of extract in mL, m = mass of extract in gram.The total content of polyphenols in the sample was expressed as % gallic acid (w / w) (Giri 2013).

TOTAL FLAVONOID CONTENT:
The Total Flavonoid Content was estimated spectrophotometrically with slight modifications, 0.1mL of a diluted extract was further diluted to a final volume of 5mL with distilled water; subsequently, 5% NaNO 2 (0.3 mL) was added, followed by 10% AlCl 3 in distilled water (3 mL) 5 min later.After 6 min, 1 M NaOH (2 mL) was added and the absorbance was measured at 415 nm.Quercetin was used as the standard for constructing a calibration curve (y = 0.0368x + 0.0017), while the flavonoid contents were expressed as mg quercetin equivalent/g of dry sample.Data were reported as the mean ± SD for duplicate measurements.(Zhishen 1999)

ANTIBACTERIAL ACTIVITY:
The antibacterial activity of Tulipa Systola ethanol extracts were examined against four strains of standard bacteria namely Escherichia coli (G-ve), Staphylococcus aureus (G+ve), Bacillus sp.(G+ve) and Pseudomonas aeruginosa (G-ve) using agar diffusion disc method.

AGAR DIFFUSION DISC:
Five millimeter filter paper discs (Whatman no. 3) were impregnated with 1 ml of each of the different plant extract dilutions (50, 100, 150 and 200 mg/mL).The discs were allowed to remain at room temperature until complete diluent achieved evaporation and kept under refrigeration until ready to be used.Discs loaded with natural products were placed onto the surface of the agar.The plates incubated for 24 hr.at 37 °C, under aerobic conditions.After incubation, confluent bacterial growth was observed.Inhibition of the bacterial growth was measured in (mm), see table (2).
The stock solution of plant extracts was prepared by adding 1 g of ethanol Tulipa Systola extracts to 5 mL of distilled water then this stock solution was sterilized by using (Millipore filters 0.2μm).The concentrations (50, 100, 150 and 200 mg/mL) prepared from the stock solution then added to 500 mL of Czapek (Dox) Agar (CDA) and poured on to sterilized Petri dishes then inoculate by bacterial strains, a sterilized Petri dish with no addition of plant extract (CDA medium only) used as control was also inoculated by bacterial strains (Mackie 1989).

RESULT AND DISCUSSION:
This is the first report on the phytochemical and antibacterial screening of Tulipa Systola growing from Kurdistan region-Iraq.The results of the study revealed that, the ethanol extracts of Tulipa Systola exhibited strong antibacterial activity against both gram positive and gram negative bacteria's, The results was taken by considering the zone of growth and inhibition of the organisms by the test fractions (Mackie 1989).
The presence of alkaloids in plants may have participated in the sequences of the plant metabolism and the presence of Terpenoids may have showed cytotoxic activity against a wide range of organisms, ranging from Bacteria and Fungi.Furthermore the presence of flavanoids and tannins in the plants may be responsible for the radical scavenging effect; flavanoids and tannins are polyphenolic compounds and are a major group of plant compounds that acts as primary antioxidant and radical scavengers.These phytochemical constituents are the key candidates in the medicinal value of the plant which will encourage researchers for further investigation and isolation of the most bioactive compounds by opportune developed techniques.Among plant antioxidant and antiradical substances in vitro, simple phenols and flavonoids are the most common components of raw extracts.Besides a significant antioxidant activity, antitumor, antiviral and antibiotic properties are frequently reported for plant which have often been identified as active principles of numerous folk herbal medicines.It was, therefore, interesting to measure the total phenolic content as well as total flavonoid content in the different extracts of T. systola to estimate their contribution to the beneficial effects of the plant.The method is based on the spectrophotometric determination of the azo-chromophore absorbance at 415 nm for total flavonoids and the phenolic contents at 760nm were shown in (Fig. 1 and 2) Interestingly, TF1 extract exhibited higher phenolic contents compared to the extracts of roots and leaves.Indeed, rather unexpected, TR1 was higher content of flavonoids recorded but among the least active extracts.While other extracts, in addition to higher phenolic contents also contribute to the higher antibacterial activity of the most active extracts, such as TF1extract.

Figure 1 :
Figure 1: Total polyphenol content of Tulipa Systola ethanol and aqueous ethanol extracts.

Figure 2 :
Figure 2: Total flavonoid content of Tulipa Systola ethanol and aqueous ethanol extracts.