Phytochemical Analysis, Antibacterial and Antioxidant Activity of the Leave Extracts of Ruta Chalepensis Antimicrobial activity, antioxidant activity, phytochemical profile, Ruta chalepensis

Rue ( Ruta chalepensis ) commonly known as rue, is traditionally used in Ethiopia for the treatment of a variety of diseases. Therefore this study conducted to investigate phytochemical constituents, antibacterial and antioxidant activity of the leave extracts of Ruta chalepensis . The phytochemical screening tests were conducted as per the standard procedure to identify the classes of compounds present in the leave extract of Ruta chalepensis . In vitro antibacterial activities of crude extracts were evaluated via disc diffusion method while antioxidant activities of extracts were determined by DPPH assay, the phosphomolybdenum method and total flavonoid contents were determined as per of the standard procedures. Phytochemical screening of leave extracts of Ruta chalepensis revealed the presence of secondary metabolites. The methanol extract had the greatest DPPH scavenging (93.851±0.148%) and total antioxidant (1.63 ± 0.19 mg AAE/g of dried extract) activities. Ethyl acetate extract showed the growth suppression of Bacillus cereus and Escherichia coli and methanol extract showed growth suppression of Bacillus cereus and Staphylococcus aureus with minimum inhibition concentration 50mg/mL. The antibacterial activities of the extracts were compared with a commercially available antibiotic (chloramphenicol) and showed moderate antibacterial activities, with inhibition zones ranging between 18–28 mm. Therefore the solvent extracts of Ruta chalepensis revealed the presence of important secondary metabolites, antibacterial and antioxidant activities. The upper layer turns red and the sulphuric layer was showed with green fluorescence. This indicates the presence of steroids. Tests for terpenoids: 1 mL of each solvent extracts were added separately to 1 mL of acetic anhydride and 1 mL of concentrated H 2 SO 4 . Formations of blue-green rings indicate the presence of terpenoids. and broth dilution methods. The antimicrobial and antioxidant property of Ruta chalepensis probably associated with the presence of alkaloids, cardiac glycosides, flavonoids, tannins, coumarins, anthraquinones, saponins, volatile oil, volatile bases, cynagenic glycosides, glucosinolates, sterols and/or triterpenes. It is concluded that this study would lead to the establishment of some valuable compound that has to be used to formulate new, different and more potent antimicrobial drugs of natural origin. Further studies are needed to identify the biologically active compounds and to evaluate the efficiency of the compound against pathogenic microorganisms associated with various human diseases.

volume of concentrated sulphuric acid was added by sides of the test tube. The upper layer turns red and the sulphuric acid layer was showed yellow with green fluorescence. This indicates the presence of steroids. Tests for terpenoids: 1 mL of each solvent extracts were added separately to 1 mL of acetic anhydride and 1 mL of concentrated H2SO4. Formations of blue-green rings indicate the presence of terpenoids.

Determination of total flavonoid content (TFC)
Total flavonoid was estimated using the method of Ebrahimzadeh et al., [9]. The extract (1 mL, 1 mg/mL) was diluted with 1.25 mL distilled water and 75 μL 5% NaNO2 was added to the mixture. After 6 min, 150 μL 10% AlCl3 was added and after another 5 min, 1 mL 1M NaOH was added to the mixture. Immediately, the absorbance of the mixture, pink in color, was determined at 510 nm versus prepared water blank. A standard curve was prepared using 5 -120 μg/mL of catechin. Results were expressed as milligram of catechin equivalents per milligram of dry extract of the plant extract. (Y = 0.022x + 0.041, R 2 = 0.99, P < 0.001).

Antioxidant activity tests DPPH assay:
The DPPH free radical scavenging activity of the extracts of leaves of Ruta chalpensis was assayed according to the method of Szollosi et al., [10], with slight modification. Different concentrations (50 to 1000µg/mL) of the extracts were taken in different test tubes. Freshly prepared DPPH solution (2 mL, 0.006%, w/v) was prepared in methanol was added in each of the test tubes containing 1 mL of the extract. The reaction mixture and the reference standards (ascorbic acid and BHT) were vortexed and left to stand at room temperature in the dark for 30 min. The absorbance of the resulting solution was then taken at 520 nm. Methanol was used as blank. The ability to scavenge the DPPH radical was calculated using the following equation: 100 ) ( (%) x Ac As Ac scavenged DPPH   Where Ac is the absorbance of the control and As is the absorbance in the presence of the sample of the extracts.

Total antioxidant using phosphomolybdenum method
The total antioxidant activities of the crude extracts were evaluated by the phosphomolybdenum method reported by Prieto et al., [11], with slight modification. 0.3 mL plant extract (0.5 and 1 mg/mL) was mixed with 3 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The samples were incubated at 95 0 C for 90 min, was cooled to room temperature and absorbance was measured at 695 nm. 3 mL of methanol was used as a blank. The total antioxidant activity was expressed as milligram ascorbic acid equivalent/gram of dried extract (mg AAE/g) (y = 0.321x + 0.019; R 2 = 0.996, P < 0.001) based on the calibration curve.

Evaluation of antibacterial activity Disc diffusion method
Antimicrobial activities of methanol, acetone, hexane and ethyl acetate extracts of Ruta chalpensis were determined by disc diffusion [12] and broth dilution methods. Culture media of mannitol salt agar for staphylococcus aureus, salmonella shigella agar for Salmonella typhi, ECD agar for E.coli and Bacillus cereus agar for bacillus cereus were prepared and poured into the plates. The depth of the medium should be 4 mm. Three to four similar colonies of pure cultures were inoculated with dextrose broth, incubated at 37 o C for 2-8 h and the size of the inoculum was adjusted to yield uniform suspension containing 105-106 cells/mL (McFarland's standard). The agar surfaces of the plates were swabbed with test culture in three directions turning the plates to 60˚ between each swabbing. Confluent growth is desirable for an accurate results. The sterile discs were (6 mm; Himedia) used for the loading crude plant extracts. Four different concentrations were prepared (100, 50, 25 and 12.5 mg/mL) and loaded in an appropriate disc. The impregnated discs were incubated at 37 o C for an hour. The dried discs were placed over the surface of the swabbed medium with equal distance to avoid the overlapping of the zone of inhibition. Pre-diffusion time was given to the swabbed plates in refrigerator condition for 5 min. The plates were incubated at 37 o C for 16-18 h during which the activity was evidenced by the presence of a zone of inhibition surrounding the discs. Each experiment was done in triplicate. A panel of antibiotics was used against each microbial strain and compared with the standard of chloramphenicol control standard. A filter paper disc impregnated with Ciprofloxacin prepared in DMSO (50 mg/mL) was used as positive control and DMSO was used as a negative control. The inhibition zone diameters were measured in millimeters.

Broth Dilution Method
The broth dilution method was used to determine the minimum inhibitory concentration (MIC) of the extracts in Dextrose Broth (Himedia-M 033) as specified by the National Committee for Clinical Laboratory Standard. A total of 10 mL of the broth was dispensed into a separate test tube and was sterilized at 121 o C for 15 min and then allowed to cool. Two-fold serial dilutions of the extracts in the broth were made from the stock concentration of the extracts to obtain 1.563-100 mg/mL for the extracts. About 0.1 mL of the standardized inoculums of the microbes were inoculated into the different concentrations of the extracts in the broth. The test tubes of the broth were incubated at 37 o C for 24 h and observed for turbidity. The lowest concentration that showed no turbidity in

RESULTS phytochemical screening
The preliminary phytochemical qualitative analysis of methanol, acetone, n-hexane and ethyl acetate extracts of Ruta chalpensis is summarized in Table 1. Terpenoids Cardiac glycosides ----6 Phenols + ---7 Saponins Tannins + ---9 Anthraquinones: The results showed the presence of alkaloids, flavonoids, anthraquinones, tannins, terpenoids, phenols and saponins in methanol extract, steroids, terpenoids and saponins were presence in ethyl acetate extract, steroids, terpenoids, saponins and anthraquinones were presence in acetone extract and flaonoids, terpenoids and anthraquinones were presence in n-hexane. Phytochemical screening test actually helps in isolating and characterizing the chemical constituents present in the plant extracts and the knowledge of the chemical constituents of plants is desirable to understand herbal drugs and their preparations and finally in discovering the actual value of folkloric remedies [13]. Phytochemicals such as alkaloids, flavonoids, steroids, terpenoids, cardiac glycosides, phenols, saponins and tannins present in different extracts exhibit a number of biological activities and protect from most of the chronic diseases [14,15]. and often used as medications and recreational drugs [16].

DPPH scavenging
The degree of discoloration of violet colour of DPPH radical, as it gets reduced, indicates the radical scavenging potential of the antioxidant [17]. The DPPH radical scavenging effects of extracts of Ruta chalpensis in different solvent systems showed in table 2. At the concentration of 1000 μg/mL used, the scavenging effect of L-ascorbic acid, BHT, and Ruta chalpensis extracts, on the DPPH radical decreased in the order of L-ascorbic acid > BHT > methanol > acetone >ethyl acetate>n-hexane, which were (97.   Table 3 followed the order: acetone > ethyl acetate > n-hexane > methanol. There was significant difference (P < 0.05) in TFC among methanol, acetone, ethyl acetate and n-hexane.     Table 4 summarizes the results of disc diffusion assays of the crude solvent extracts of Ruta chalpensis. All result showed that the extracts possess antimicrobial activities against the selected microorganisms. The antimicrobial activities of the extracts (methanol, acetone, hexane and ethyl acetate) at different concentrations were screened by the disc diffusion method and the mean value of zone of inhibition was assessed in millimeter diameter. The results are given in the tables-4. In the disc diffusion method all extracts showed ≥ 11 mm mean zone of inhibition, further those microorganisms were tested for MIC by broth dilution technique the results are given in tables-5. The result revealed that all extract 25mg/mL was obtained as MIC value against all microorganisms. The activity somehow nearer to zones produced by the control antibiotics (chloroaphenol).

DISCUSSION
Now a days there has been considerable concern in the use of plant material as an alternative drug [18] and many components of plant products have been shown to be specially targeted against resistant pathogenic bacteria [19]. Multidrug resistant strain of many pathogens is a serious threat and makes chemotherapy more difficult. The toxicity of new generation antibiotic drugs discourages their use in treatment. Moreover, the current cost of most of the chemotherapeutic agents is unbearable to the public especially in developing countries [20]. The present work was a pioneer attempt to investigate the phytochemical screening, antimicrobial and antioxidant properties of Ruta chalepensis.
Flavonoids are the polyphenolic compounds in the human diet and are found in all plants. The pharmacological effects of flavonoids include CNS activity, cardiotonic, lipid lowering, antiulcer, hepatoprotective, anti-inflammatory, antineoplastic, antimicrobial, antioxidant and hypoglycemic activity. Dietary intake of flavonoids containing foods potentially higher in antioxidant activities [21]. Steroids and triterpenoids are pharmalogically active compounds and show the analgesic properties [22]. The steroids also exhibit central nervous system activities. [23] reported the terpenoids to decrease blood sugar level in animals. Cardiac glycosides are also of medicinal importance and used in the treatment of congestive heart failure and cardiac arrhythmia [24]. Phenols and phenolic compounds have tremendous antimicrobial potential. They have been extensively used in disinfections and remained the standards with which other bactericides are compared [25]. They have been reported to exhibit cellular defense mechanism in atherogenesis and cancer. A wide range of phenolic substances show strong antioxidant and antimutagenic activities. As per recent evidences, phenolic compounds could also play an essential health promoting role [26]. Saponins are being promoted commercially as dietary supplements and nutraceuticals in traditional medicine preparations [27]. They also possess hypocholesterolemic and antidiabetic properties [28]. Certain tannins (ellagitannins from Lagerstroemia speciosa) stimulate glucose uptake. They exhibit insulin like activity acting as glucose transport activators of fat cells [29].
The ethyl acetate extract of the leave of Ruta chalepensis possessed comparatively highest antibacterial activities for Bacillus cereus (17.00 mm) and Escherichia coli (15.67 mm) and methanol extract possessed highest antioxidant activities (93.851%). The results of other extracts also showed considerable antimicrobial activity against bacteria in disc diffusion and broth dilution methods. The antimicrobial and antioxidant property of Ruta chalepensis probably associated with the presence of alkaloids, cardiac glycosides, flavonoids, tannins, coumarins, anthraquinones, saponins, volatile oil, volatile bases, cynagenic glycosides, glucosinolates, sterols and/or triterpenes. It is concluded that this study would lead to the establishment of some valuable compound that has to be used to formulate new, different and more potent antimicrobial drugs of natural origin. Further studies are needed to identify the biologically active compounds and to evaluate the efficiency of the compound against pathogenic microorganisms associated with various human diseases.

CONCLUSIONS
In this work the phytochemical screening, antioxidant and antibacterial activity of the leaves extract of Ruta chalepensis ware investigated. The phytochemical screening of different solvent extracts of leaves of plant of Ruta chalepensis revealed the presence of important secondary metabolites in the leaves. The ethyl acetate extract of the leave of Ruta chalepensis possessed comparatively highest antibacterial activities for Bacillus cereus and Escherichia coli and methanol extract possessed highest antioxidant activities. The presence of phytochemicals can make Ruta chalepensis, a potential drug. However, further study is necessary to quantify, isolate, characterize and other biological activity of the particular compound for drug development.