HOW FAR WOULD DIFFERENT SORTS OF FERTILIZERS AFFECT THE PRODUCTIVITY OF RUE (RUTA GRAVEOLENS L.) M.Y. Khalil and N.Y.

Naguib Cultivation and Production of Medicinal and Aromatic Plants. NRC. Cairo, Egypt

Abstract: Two seasons pots trials were carried out in the open field at the Experimental Farm of National Research Centre, Dokki, Egypt, During 2005/2006 and 2006/2007, in order to investigate the impact of yeast, EM, Azotobacter (A. chroococcum), provided from Rhizobacterin, (107 – 108 cfu/g) at the rate of 2 g/L, compost at the rate of 1 kg/pot and 2 g NPK (2: 1: 1) fertilizers, individually or combined with half the dose of NPK, on vegetative growth, flowers yield, oil production and oil constituents, phenolic compounds and antioxidant activity in rue (Ruta graveolens L.). The obtained resulted revealed that fertilization treatments significantly stimulated most of the studied characters compared to the control. Yeast combined with half dose of NPK significantly resulted the highest values of plant height, fresh and dry weight of herb and flowers per plant and feddan (an Egyptian measure of area, equals 4200 m2), oil percent and oil yield of herb and flowers. The major components of phenolic compound in herb or flowers were: rutin, salycilic acid, ferulic acid, caffic acid pyrogallic acid, amounting to 89.1 and 77.8% of phenolic compounds in herb and flowers, respectively. Yeast combined with NPK gave the highest antioxidant activity at 100 µl extractions in both herb and flowers, while compost plus NPK and azotobacter plus NPK led to the highest antioxidant activity in herb and flowers, respectively at 150 or 200 µl extractions. EM + NPK resulted the highest percentage of rutin or cumarin in rue leaves the yeast combined with NPK treatment produced the largest production of the two compounds/ plant, as compared to other fertilizer treatments, since yeast + NPK gave the highest yield of herb. Twenty eight compounds were detected in herb oil, comprised 98.26% of the oil, seven of them was hydrocarbons but 21 were oxygenated. Whereas in the oil of flowers, 25 compounds were detected constituting 99.40%, nine of them were hydrocarbons against 16 oxygenated. The major constuents of the oils, in descending order were: 2-Undecanone, 1-Undecene and 2-Nonene. They amounted to about 78.4 and 80.9% of herb and flowers oil, consecutively. However, the 2-Undecanone compound for more than half of the oils. Keywords: rue (Ruta graveolens L.); Yeast; EM; Azotobacter. INTRODUCTION Rue (Ruta graveolens L.), a member of family rutaceae, is an annual medicinal herb, native to the temperate zone (Bradly, 1998). The genus Ruta acquired its same from its main constituent rutin, responsible for bitter taste of rue, a polyphenolic flavonole glycoside containing the disaccharide rutinase as sugar component (Migud, 2003 and Kugovkina et al, 2004). It has a widespread reputation as a popular folk medicine in various countries. In China, the leaves are applied to reduce inflammation from snake bites, insect bites, strains and sprains and antispasmodic for smooth muscles. Unani medicine of India recommends rue to treat various physical conditions and to improve mental clarity

and as an aphrodisiac. In Latin America rue is well-known as cold, menstrual cramp remedy, where an ointment is applied for gout and rheumatic pains, and strong rue tea compresses are placed on the chest for bronchitis, and it is used to kill intestine parasites. Arabs add rue to suspect water to counteract any ill effect in South Africa, women use rue to promote menstruation (Pino et al, 1997; Atta and Alkofahi, 1980; Miguel, 2003; Chavez et al, 2003 and Steenkampt, 2003). Recent research works indicated many beneficial uses of rue. The medicinal action of common rue would be abortificient, anthilimintic, antiseptic, antispasmodic, carminative, irritant and stomachic. Rue was once officially recognized treatment for hypertension, diabetes and allergic reaction (Browner, 1985). Atta and Alkofahi (1980) and Chavez et al, 2003 reported that main uses were to relive gaiety and rheumatic pains and to treat nervous heart problems. Chiu and Fung (1997) indicated that rue contained cardiac vascular active substances that had a direct effect on cardiovascular system. Miguel (2003) concluded that rue oil was a central nervous system depressant and at high doses a narcotic poison and eliminate worms. Pathak et al (2003) found that rue in combination with Co3(PO4)2 could be used for treating brain cancers particularly glioma. Lauk et al (2004) confirmed the anti-inflammatory properties of rue. Not only human effect, but also rue extract had an inhibitory effect on vitro germination of radish seeds a radical growth (Aliotta et al, 1994 and 1996) and several seeds (Vincenzo et al, 2002). Plant growth and production are greatly affected by nutrient supply: organic and biofertilizers are recently recommended to ensure safety for human health as well as the surrounding environment (Abd El-Gawad, 1999). Active dry yeast is a natural safety biofertilizer that is rich in protein, Bvitamins and natural plant growth substances as cytokinins. It also releases CO2 that improve net photosynthesis. Many researchers gained good growth flowering and production of several plants when applied yeast, as Naguib and Khalil (2002) on black cumin, and El-Sherbeny et al (2007a) on Ruta graveolens. Azotobacter chroococcum bacteria are famous nitrogen fixer that had beneficial effect on different plants as Agamy (2004) on fennel and Khalil (2006) on Plantago afra. Compost provide show release of nutrients, improve soil structure, aeration, water holding capacity and availability of soil micro-organisms (Herrera et al, 1997). Many investigators had good results when using compost for Tagetes erecta (Khalil et al, 2002), German chamomile (Naguib, 2003), Sideritis montana (El-Sherbeny et al, 2005) and Ruta graveolens El-Sherbeny et al (2007b) The present work was designated to find out the effect of different mineral, organic and biofertilizers on growth, flowering, oil production and chemical constituents of rue (Ruta graveolens L.) under Egyptian condition. Material and Methods Seeds of rue (Ruta graveolens L.) supplied from Experimental Farm of Pharmaceutical Science Dept. at Giza were sown in 30cm clay pots at September, 20th, 2005 and 2006, in the open air outside the green house at the Experimental Farm of National Research Centre, Dokki, Egypt, to investigate the influence of yeast, EM and Azotobacter at the rate of 2.0 g/L, compost at the rate of 1.0 g/pot and NPK (2: 1:1) at the rate of 2.0 g/pot, individual or combined with half the dose of NPK. Physiochemical properties of the used soil were determined using the procedures by Klute (1986) and presented in Table (a).

Physical-chemical properties of experimental soil: Texture Coarse sand (%) Fine sand (%) Loamy 8.7 30.0
EC (mmhos/cm)

Silt (%)

Clay (%)

pH

29

30.0
Fe 3.0 Zn 4.5 Mn 2.0

7.8
Cu 0.80

6.5

N 14.0

P 0.54

K 3.0

S 4.2

Na 8.0

Ca (ppm) 9.2

Mg 14

Yeast was dissolved in distilled water and activated with a spoonful of black honey. Compost was a product of El-Obor Company, having the following properties in Table (b). Physical-chemical properties of the organic compost:
Moisture % 27-46 pH 7.2-7.6 EC 3-4 Total (N%) 1.4-1.7 Organic matter % 38-50 Ash % 41-43 C/N ratio 13.6:1 P % 0.47 K % 1.2-1.6 Fe ppm 800 Mn ppm 190 Cu ppm 40
7

Zn ppm 70

Azotobacter chroococcum bacteria was provided from Rhizobacterin (10 – 108 cfu/g) obtained from Agricultural Microbiology Dept., Agriculture Research Center, (ARC) as a peat-based inoculums. Azotobacter or EM were inculcated to the seeds using aqueous solution of arabic gum as an adhesive, then the seeds were dried in shade as suggested by Barakat et al (2004). NPK treatment was supplied from ammonium sulphate (21.5% N), calcium superphosphate (15.5% P2O5) and potassium sulphate (48.0% K2O) to achieve the ratio of 2: 1:1. The fertilizers were applied at two times, the first one month after sowing and the other one month later. The study comprised 10 treatments as the following: control, yeast, EM, azotobacter, compost, NPK, yeast + ½ dose of NPK, EM + ½ dose of NPK, Azotobacter + ½ dose of NPK and compost + ½ dose of NPK. Treatments were replicated three times. Before sowing each pot was supplied with 2.0 g of calcium super phosphate (15.5% P2O5) and potassium sulphate (48.0% K2O). All routine agriculture practices as watering, weeding, pest control………..etc were conducted whenever required. At the full flowering stage, which occurred at 25th May, 2006 in the first season and 30th May, 2007 in the second ones plants were collected and the following data were recorded: 1-Vegetative growth characters: Plant height (cm), number of branches/plant, fresh and dry weight of herb and flowers (g/plant) and yield per feddan was assessed (Feddan: is an Egyptian measure for area that equals 4200 m2). 2- Essential oil production: The essential oil of fresh herb and flowers was extracted from each treatment, by hydrodistillation using Clevenger-type apparatus according to AOAC (1995). - Percentage of oil (v/w): was calculated based on herb or flower fresh weight. - Oil yield: ml/plant and kg/feddan were calculated. 3- Essential oil components: Oil samples for the treatments were analyzed by GLC apparatus. Separation of the resulted volatile oil was accomplished on a Varian Gas Chromatography (Thermo Inst., USA) mass spectrometer and a 30 cm x 0.25 mm. DB-5 capillary column film thickness (J & W Scientific, USA). The column temperature was programmed from 50 0 C (constant for 3 min.) at a rat of 7 0C / min to 250 0C with 10 min. isothermal hold. The injector temperature was 2200 and transition time temperature was 250 0C. The carrier gas was helium and the column head pressure was 10 – 15 psi. The identification of the constituents was determined by comparing the spectrum with the

other stored in Wiley Mass Spectral Library containing over 147000 volatile compounds. 4- Phenolic compounds: Samples of the collected fresh herb and flowers of rue (Ruta graveolens L.) were cleaned and immediately stored at –20oC until lyophilized (Delta, condenser temperature -45oC pressure 0.01 m bar). After lyophilization, the freeze-dried tissues were ground to pass a 0.5 mm sieve and allowed to equilibrate in open air. The phenolic compounds were extracted according to Hertog et al. (1992). Extracts were prepared as follows: 40 ml of 62.5% aqueous methanol [2 g/L of antioxidant tert. Butylhyroquinone (TBHQ)] was added to 0.5 g of freeze-dried sample material. 10ml of 6 M HCl was added to this extract with careful mixing. The extraction solution thus obtained consisted of 1.2 M HCl in 50% aqueous methanol (v/v). After refluxing at 90 o C for 2h with regular swirling, the extract was allowed to cool and was subsequently made up to 100ml with methanol and sonicated for 5 min. approximately 2ml was filtered through a 0.45- μm filter for organic solvents prior to injection. Identification of individual phenolic compounds of the plant extracts was performed on a Hewlett-Packard HPLC (Model 1100). Using a hypersil C18 reversed-phase column (250 x 4.6 mm) with 5 urn particle size. Injection by means of a Rheodyne injection valve (Model 7125). A constant flow rate of 1 ml min’1 was used with two mobile phases: (A) 0.5% acetic acid in distilled water at pH 2.65; and solvent (B) 0.5% acetic acid in 99.5% acetonitrile. The elution gradient was linear starting with (A) and ending with (B) over 35 min, using an UV detector set at wavelength 254 nm (BenHammouda et al. 1995). Phenolic compounds of each sample were identified by comparing their relative retention times with those of the standards mixture chromatogram. The concentration of an individual compound was calculated on the basis of peak area measurements, and then converted to mg phenolic/100g dry weight. All chemical and solvents used were HPLC spectral grade. Standard phenolic compounds were obtained from Sigma (St. Louis, USA) and Rom Merck-Shcuchrdt (Munich, Germany Chemical Companies). 5- Antioxidant activity in a linoleic acid system: Antioxidant activity assay was carried out by using the linoleic acid system. Linoleic acid emulsion (0.02 M) was prepared with linoleic acid (0.2804 mg) and Tween 20 (0.2804 g) in phosphate buffer (50 ml, 0.05 M, pH 7.4). A reaction solution, containing extracts (50 µl – 200 µl). Linoleic acid emulsion (2.5 ml), and phosphate buffer (2.3 ml, 0.2 m, pH 7.0) were mixed with a homogenizer. The reaction mixture was incubated at 37 oC in the dark, and the degree of oxidation was measured according to the thiocyanate method (0.1 ml, 30%), and sample solution (0.1 ml). After the mixture was stirred for 3 min, the peroxide value was determined by reading the absorbance at 500 nm, and the inhibition percentage of linoleic acid peroxidation was calculated as (%) inhibition = [ 1- (absorbance of sample at 500 nm) / (absorbance of control at 500 nm)] x 100. All tests were run in duplicate, and analysis of all samples was done in triplicate. 6- Rutin and Coumarin percentage: The percentage of rutin and coumarin were determined in dried leaves using the methods prescribed by Zhuang et al (1992) and Harbone (1998), respectively. Statistical analysis: the layout of the experiment was complete randomized blocks with three replications. Analysis of variance was done according to Snedecor and Cochran (1990), Comparison between treatments was carried out by L.S.D. at 0.05 level.

RESULTS AND DISCUSSION
Vegetative growth parameters: Data in Table (1) indicated that all fertilization treatments significantly improved plant height (cm) and herb fresh and dry weight (g) of rue (Ruta graveolens L.) plants, in comparison with control. The findings coincide with those obtained by many researchers on various plants: Khalil (2002) on Rosmarinus officinalis, Naguib (2003) on Chamomilla racutita, Naguib et al (2003) on some radish cultivars, El-Sherbeny et al (2005) on Sideritis montana and El-Sherbeny et al (2007a, b and c) on Ruta graveolens L. Table (1): Effect of various fertilizer treatments on plant height, number of branches, herb fresh and dry weight of Ruta graveolens plant, during two seasons 2005 / 2006 and 2006 / 2007 Characters Plant Branches Herb fresh weight Herb dry weight height No /plant g/plant Kg/fed. g/plant Kg/fed. (cm) Treatments First season 2005 /2006 Control 32.60 6.00 68.79 1031.85 15.82 237.30 Yeast 38.10 7.33 109.68 1645.20 25.77 386.55 EM 35.70 6.30 92.43 1386.45 21.33 319.95 Azotobacter 36.00 6.50 100.67 1510.05 23.67 355.05 Compost 34.50 6.22 89.92 1348.80 20.72 310.80 NPK 37.67 7.33 106.17 1592.55 24.95 374.25 Yeast + ½ NPK 46.10 9.21 155.50 2332.50 36.54 548.10 EM + ½ NPK 43.67 8.73 139.67 2095.05 32.82 492.30 Azotobacter + ½NPK 44.72 8.94 143.33 2149.95 33.50 502.50 Compost + ½NPK 43.08 8.61 120.33 1804.95 28.28 424.20 LSD at 5% level 1.02 N.S. 3.11 16.33 2.01 11.33 Second season 2006 / 2007 Control 31.00 5.00 69.51 1042.65 16.20 243.00 Yeast 39.33 6.73 112.33 1684.95 25.85 387.75 EM 37.00 5.65 93.07 1396.05 21.69 325.36 Azotobacter 37.78 5.68 102.00 1530.00 23.46 315.90 Compost 36.25 5.56 87.18 1307.70 20.32 304.80 NPK 38.12 6.00 107.50 1612.50 25.05 375.75 Yeast + ½ NPK 48.33 8.00 143.50 2152.50 33.14 497.10 EM + ½ NPK 44.50 7.00 120.33 1804.95 27.80 417.00 Azotobacter + ½NPK 46.67 7.33 126.67 1900.05 29.25 438.75 Compost + ½NPK 42.50 6.80 117.67 1765.05 27.09 406.35 LSD at 5% level 1.12 N.S. 2.30 12.50 1.01 12.37 The findings of Naguib and Khalil (2002) on black cumin, El-Ghadban et al (2003) on Castor oil and Heikal (2005) on thyme. Flowers fresh and dry weight (g): It is clear from data in Table (2) that ruta flowers fresh and dry weight (g) per plant or kg/feddan attained a similar trend to that of the vegetative growth characters. All fertilizers under study significantly increased such characters as compared to the

control, at both seasons. Adding have the dose of NPK to other fertilizers significantly increased flowers weight as compared with individual fertilizers, during both seasons. Meanwhile, the yeast + NPK treatment resulted significantly the heaviest weights (14.33 and 10.66g) flower fresh weight and 1.86 and 1.39 g dry weight/plant in the first and second seasons, consecutively), as compared to 5.23, 0.68 and 0.50g for both parameters of the control, at the two seasons, successively. Then, came the azotobacter + NPK treatment, which led to significant increases compared to most treatments. In this concern, El-Sherbeny et al (2007a) claimed that fertilization promoted flowers yield of Ruta graveolens L., a similar result to that on the second work. Table (2): Effect of some fertilizer treatments on fresh and dry weight of flowers of Ruta graveolens plant during two seasons 2005/2006 and 2006/2007. Characters Flowers fresh weight Flowers dry weight Treatments Control Yeast EM Azotobacter Compost NPK Yeast + ½ NPK EM + ½ NPK Azotobacter + ½NPK Compost + ½NPK LSD at 5% level g/plant 5.23 10.33 7.29 8.49 6.67 9.14 14.33 12.64 13.00 11.98 1.18 Kg / fed. g/plant Kg / fed. 10.20 20.14 14.20 16.56 13.01 17.82 27.94 24.65 25.35 23.36 1.30 First season 2005 / 2006 78.45 0.68 154.95 1.34 109.20 0.95 127.35 1.10 100.05 0.87 137.10 1.19 214.95 1.86 189.60 1.64 195.00 1.69 179.70 1.58 12.10 0.23 Second season 2006 / 2007 58.05 0.50 116.40 1.01 77.54 0.67 80.86 0.70 71.25 0.62 94.20 0.82 159.90 1.39 131.25 1.14 140.25 1.22 121.95 1.06 10.52 0.30

Control 3.86 7.50 Yeast 7.76 13.13 EM 5.17 10.08 Azotobacter 5.39 9.10 Compost 4.75 9.30 NPK 6.28 12.25 Yeast + ½ NPK 10.66 20.85 EM + ½ NPK 8.75 17.10 Azotobacter + ½NPK 9.35 18.30 Compost + ½NPK 8.13 15.90 LSD at 5% level 2.50 1.51 Essential oil production: It is appeared from Table (3) that flowers had higher oil percentage the herb, but as weight of the herb was largely greater than the weight of flower in plant, so the yield of herb oil was greater than flowers oil. However, oil production in both parts attained a parallel trend. All fertilizers treatments (individual or combined with NPK) significantly raised essential oil production, represented by oil percentage and oil yield per plant (ml) and per feddan (L), in the herb and flowers of ruta plant, as compared to the control (unfertilized treatment), at both seasons. Such increase over control ranged

between 0.005 and 0.025%, 0.020 and 0.145 ml/plant and 0.285 and 2.188 L/feddan for herb oil and 0.127%, 0.003 and 0.029 ml/plant and 0.052 and 0.448 L/feddan for flowers oil in the first season. Results of the second season showed similar trend. For the fertilizers, yeast had a favourable effect than the individual fertilizers. Adding half dose of NPK to different fertilizers significantly increased oil production as compared with individual ones. Yet, the impact of yeast combined with NPK was sound for various oil production parameters. This treatment produced 0.127%, 0.197ml/plant and 2.962 L/feddan for herb oil against 0.252%, 0.03 ml/plant and 0.54 L/feddan for flowers oil, at the first season; while in the second season the productions were 0.109, 0.156, 2.346, 0.255, 0.027 and 0.408 for the aforesaid characters, respectively. Such findings would be reasonable, since both vegetative growth anf flower fresh and dry weight attained parallel trend. Table (3): Effect of some fertilizer treatments on essential oil of Ruta plant two seasons 2005/2006 and 2006/2007. Characters Essential oil (herb) Essential oil (flowers) % ml/plant L/fed. % ml/plant L/fed. Treatments First season 2005 / 2006 Control 0.075 0.052 0.774 0.125 0.0066 0.098 Yeast 0.096 0.105 1.579 0.188 0.019 0.285 EM 0.084 0.077 1.165 0.150 0.011 0.165 Azotobacter 0.085 0.086 1.284 0.155 0.013 0.195 Compost 0.080 0.072 1.079 0.144 0.010 0.150 NPK 0.092 0.098 1.465 0.176 0.016 0.240 Yeast + ½ NPK 0.127 0.198 2.962 0.252 0.036 0.540 EM + ½ NPK 0.106 0.148 2.221 0.216 0.027 0.405 Azotobacter + ½NPK 0.114 0.163 2.461 0.223 0.029 0.435 Compost + ½NPK 0.112 0.135 1.841 0.198 0.034 0.360 LSD at 5% level 0.006 0.009 0.011 0.009 0.005 Second season 2006 / 2007 Control 0.063 0.044 0.659 0.147 0.0067 0.085 Yeast 0.093 0.104 1.567 0.200 0.0155 0.233 EM 0.087 0.081 1.215 0.188 0.0097 0.146 Azotobacter 0.093 0.095 1.423 0.194 0.010 0.157 Compost 0.086 0.075 1.125 0.185 0.0088 0.132 NPK 0.089 0.096 1.435 0.193 0.012 0.180 Yeast + ½ NPK 0.109 0.156 2.346 0.255 0.027 0.406 EM + ½ NPK 0.102 0.123 1.841 0.219 0.019 0.287 Azotobacter + ½NPK 0.104 0.132 1.976 0.228 0.021 0.320 Compost + ½NPK 0.100 0.118 1.765 0.218 0.0177 0.266 LSD at 5% level 0.005 0.010 0.012 0.005 0.003 This results are in coincidence with those previously gained by many researchers on various plants. El-Desuky et al (2001) on sweet fennel, El-Khawwas (2002) on Nigella sativa; Naguib (2003) on Chamomilla racutita, Khalil and ElSherbeny (2005) on three mint species, and El-Sherbeny et al (2007a, b) on Ruta graveolens showed that fertilization stimulated oil synthesis through promoting the physiological processes of biochemical synthesis. The beneficial effects of yeast were attributed to its effect on carbohydrate accumulation (Winkler, 1962). Robinson

(1973) reported that yeast contained vitamins which are recognized as co-enzymes involved in oxidative and non-oxidative carboxylation processes. The biochemical active pyrophosphates are the units which condense the formation of many varied forms that constitute the terpenes. Phenolic compounds concentration: Phenolic compounds are of considerable interest from the viewpoint of dietary supplementation and food preservation (Yoshida et al, 1993). It appeared from the foregoing results on vegetative growth and flowers parameters, that the combined treatments of different individual fertilizers and half dose of NPK significantly stimulated such parameters compared to the individual fertilizers. So, the concentrations of phenolic compounds (mg/100g D.W.) were determined in ruta herb and flowers of the combined fertilization as well as the control. The results of estimation were shown in Table (4). Such data revealed that seventeen phenolic compounds were detected in ruta herb and flowers. It is evident that the five compounds: rutin, salicylic acid, ferulic acid caffic acid and pyrogallic acid composed about 89 and 77% of phenolics in herb and flowers consecutively. Yet, rutin represented the highest concentration in most treatments. Whilst, the compounds with the lowest concentrations were catechol and protocatechenic. It is clear that fertilization increased most of phenolic compounds. Compared to the control. However, the effect varied from treatment to another. Azotobacter plus NPK treatment raised rutin, ferulic acid, caffic acid, pyrogallic and catechol in herb and pyrogallic acid, catechol and protocatechenic in flowers. But, EM combined with NPK increased salicylic acid in herb and rutin, salicylic acid and caffic acid in flowers, compared with other fertilization treatment. Moustafa et al (2005) noted that the content of phenols in Nerium oleander L. leaves were basically depending on the concentration of nitrogen fertilizer. Khalil et al (2007) concluded that salicylic and pyrogallic acids were the major phenolic compounds in eight medicinal plants namely: marigold, dragonhead, fennel, plantain, clary, sage and sideritis; while catechol, Protochatechenic and cinnamic acid were found
in minor quantities.

Antioxidant activity: Antioxidants are vital substances which possess the ability to protect human from damage caused by free radical induced oxidative stress (Souri et al 2004). Natural antioxidants are usually phenolic and polyphenolic compounds including flavonoides (Velioglue et al 1988 and Kim et al, 1997). It appeared from data in Table (5) that most fertilization treatments significantly increased antioxidant activity of rue (Ruta graveolens L.) herb and flowers expressed as inhibition of peroxidation percentage, in comparison to the control (non fertilization). Increasing the concentration of ethanol (100, 150 and 200 µl extract progressively raised percentage of antioxidant activity, in most cases. In general, it was observed that antioxidant activity of rue flowers was markedly larger than that of the herb. The yeast combined with half dose of NPK resulted the significantly highest antioxidant activity in both herb and flowers at the 100µl ethanol extract. Whereas, at 150 and 200 µl ethanol extract, the compost plus NPK treatment significantly produced the largest antioxidant activity in the herb, but in flowers azotobacter combined with NPK resulted the highest percent of peroxidation inhibition, as compared with other fertilizers.

Procsfos et al (2005) on some Greek aromatic plants observed that phenolic fraction of plant extract has been linked to their antioxidant capacity and antimicrobal activity. Meanwhile, Mostafa et al (2005) claimed that antioxidant activity was the lowest in plants of the control compared to fertilized ones. Furthermore, Khalil et al (2007) reported that antioxidant activity of eight medicinal plants raised as ethanol extract was increased from 100 to 150 to 200µl extract. Table (4): The concentrations of phenolic compounds in of Ruta graveolens L. herbs at their vegetative stage (mg/100g D.W.) Phenolic Herbs Flowers compounds 1 2 3 4 5 1 2 3 4 5 112.13 80.23 101.24 130.10 145.0 33.18 66.97 44.63 82.21 25.90 Pyrogallic acid Hydroquinone 15.94 8.90 27.48 47.22 11.27 8.96 22.18 10.12 36.00 30.15 Resorcinol 32.06 28.36 25.14 9.18 19.99 27.00 12.33 24.50 15.15 9.67 Protochatechenic 2.19 -1.96 0.08 1.70 3.41 2.14 1.06 5.91 4.44 Catechol 0.52 0.26 0.45 2.94 1.03 0.65 0.85 1.12 2.50 1.29 Hydroxyl benzoic 19.77 43.25 41.37 33.90 20.44 10.84 3.92 1.25 5.34 10.10 Chlorogonic acid 2.65 10.10 21.11 17.27 9.91 5.90 17.29 7.28 12.13 5.98 Phenol 96.25 89.55 70.43 54.32 66.93 6.39 22.20 19.04 16.48 13.28 Vanillin 20.22 27.14 16.85 13.88 22.16 9.75 20.55 15.13 12.86 18.00 p-coumric acid 11.21 33.00 13.44 26.25 19.18 4.33 19.00 30.11 17.69 21.24 Ferulic acid 136.48 160.20 150.25 184.47 121.26 208.10 180.22 198.74 169.25 211.25 176.22 155.27 236.14 223.14 166.27 118.01 93.65 175.30 100.35 92.83 Salicylic acid Rutin 177.65 213.33 199.22 243.59 231.11 166.24 253.23 259.44 239.71 229.29 O-coumaric acid 16.83 47.23 27.61 34.15 24.76 5.08 8.04 16.32 7.92 10.73 Coumrin 32.66 22.91 45.90 29.68 22.22 19.32 22.73 17.25 10.34 15.56 25.90 11.65 Cinnamic acid 12.41 24.10 18.41 30.12 27.20 12.12 8.51 6.60 Caffic acid 115.24 120.35 110.18 146.88 137.90 108.15 141.43 200.00 130.25 150.22 1 = Control 2 = Yeast + NPK 3 = EM + NPK 4 = Azotobacter + NPK 5 = Compost + NPK Table (5): Antioxidant activity of ethanol extracted Ruta graveolens L. flowering stage (mg/100g D.W.) (%) Inhibition of peroxidation µl extract 100 µl extract 150 µl extract 200 Control 56.91 ± 3.25 65.33 ± 2.21 72.15 ± 2.34 Yeast + NPK 82.45 ± 1.36 77.90 ± 3.47 80.88 ± 3.08 EM + NPK 80.66 ± 2.10 83.50 ± 3.51 89.22 ± 3.47 Azotobacter + NPK 70.58 ± 3.24 81.91 ± 3.84 90.30 ± 3.11 Compost +NPK 68.52 ± 3.61 89.04 ± 3.41 92.35 ± 3.02 Control 75.17 ± 2.98 71.58 ± 3.99 82.48 ± 3.22 Yeast + NPK 88.24 ± 1.41 92.13 ± 2.81 79.83 ± 3.10 EM + NPK 72.39 ± 1.57 88.24 ± 2.35 93.28 ± 2.75 Azotobacter + NPK 2.64 ± 73.80 2.05 ± 95.91 2.92 ± 98.25 Compost +NPK 1.33 ± 81.15 1.14 ± 90.22 2.30 ± 93.66 L.S.D. at 0.05 2.47 3.65 3.45 - Each value is expressed as mean ± SE (n = 3) Flowers Herb

Essential oil components: Data in Table (6 and 7) reveled that 28 components were detected in rue herb oil, representing in average about 98.26%. From these components, seven hydrocarbonic compounds were representing about 20.05% against 21 oxygenated ones comprising about 78.21% of herb oil. Whereas, in flowers oil, about 25 compounds comprising in average about 99.40% of the oil, nine of them were hydrocarbons, constituting only 14.61% against 16 oxygenated compounds representing about 84.78%. In this respect, Pino (1997) and El-Sherbeny et al (2007a,b) could identify 2833 compounds in rue oil. The missed compounds in herb oil compared to flowers oil were four ones, namely: 2-nonene, pentacosane, cyclo-tridecanone and cyclo-undecanone. While flowers oil lacked seven compounds, all of them oxygenated, namely: 2-octanone, tetradecanol, dodecanol, neptalactol, oscoridole, hexadecanal and 3-thoxy-4 hydroxy4-(4-methoxy-phenyl) cyclopent-2-enone. Different fertilization treatment did not affect number of detected compounds except the azotobacter + NPK treatment which caused lack of 3-thoxy-4 hydroxy-(4methoxy-phenyl) cyclopent-2-enone. Such treatment led to the lowest percentage of identified componends (95.98 and 98.99%) for herb and flower oils, respectively, against 97.77 and 99.69%, consecutively, for the control. The major hydrocarbons was undecone followed by nonene and limonene which constituented about 12.64, 2.43 and 2.12%, respectively in herb oil against 4.72, 3.36 and 1.99% in flowers oil, successively. While, the lowest hydrocarbons were neophytadiene and 3,4-dihydrbenzo[b]fluoranthene (0.08 and 0.51%, respectively in
herb oil anthracene (0.27 and 0.35%, consecutively) in flowers oil. The major oxygenated constituents were: 2-undecanone and 2-nonanone which comprised about 53.33 and 12.46%, respectively, in herb oil against 66.87 and 9.35%, successively, in flowers oil. While the lowest oxygenated compounds were: 3-thoxy-4

hydroxy-(4-methoxy-phenyl) cyclopent-2-enone, hexadecanal and 1-dodecanol, 3, 7, 11-trimethyl comprising 0.09, 0.11 and 0.15%, consecutively, in herb oil. But, in flowers oil, the hexadecanoic acid (0.05%), 2 tetradecanone (0.06% and cycloundecanone (0.07%) were lowest oxygenated compounds. The recent finding are in a parallel trend with that Pino (1997), Stashenko et al (2000); Foe et al (2002) Khalid et al (2007) and El-Sherbeny et al (2007a,b and c) that 2-undecanone flowed by nonene were the major constituents of rue herb and flowers oil. Rutin and cumarin: Ruta sp. Plants acquired its generic name from its bioflavonoids rutin (Bailey, 1976). Rutin is responsible for the bitter taste of fresh rue plants. The action of rue on hypertension, diabetes and allergic reactions was attributed to alkaloids as coumarin and rutin. Rutin strengthens fragile blood vessels and helps alleviate varicose veins (Blumenthal, 2001 and Chavez et al, 2003). Data in Table (8) indicated that both rutin and cumarin (%) and content in the leaves of Ruta graveolens, were increased by fertilization treatments compared to the control. However, percentage of rutin or cumarin was insignificantly affected by various fertilizers treatments. Although largest percentage of rutin occurred due to EM+NPK treatment at both seasons, the yeast combined with NPK produced the largest content of rutin

(0.30 and 0.22 ml/plant, in both seasons, respectively) and cumarin (0.0052 and 0.0042 ml/plant, at the two seasons, successively). Such contents were significantly larger than most other treatments. This increment would be attributed to the heavy herb dry weight which was significantly heavier due to that treatment in comparison to other ones. These results are in coincidence with the finding of El-Sherbeny et al (2007a, b), that fertilization increase rutin and cumarin content in Ruta graveolenus. Table (6): Effect of some fertilizer treatments on herb essential oil constituents of Ruta graveolenus L. plants 1 2 3 4 5 Hydrocarbon compound Limonene 2.00 2.25 4.12 1.22 1.03 Geyrene 1.17 0.81 1.05 1.05 1.00 Nonene 1.90 2.55 3.52 3.32 0.88 Undecene 14.00 10.15 9.60 19.76 9.68 Anthracene 1.62 2.00 1.00 0.88 0.75 Neophytadiene 0.11 0.03 0.03 0.18 0.05 3,4-Dihydrobenzo[b]fluoranthene 0.33 1.12 0.78 0.11 0.20 Total hydrocarbon 21.13 18.91 20.10 26.52 13.59 Oxygenated compound 2-Octanone 0.30 0.26 0.11 0.27 0.12 2-Nonanone 20.00 16.11 10.60 6.37 13.23 Teteradecanal 0.18 1.00 0.51 0.92 1.96 Dodecanal 0.95 1.00 1.19 0.40 0.58 2-Docanone 0.66 0.27 1.88 0.09 0.31 2-Undecanone 49.44 51.15 55.90 53.05 57.09 2-dodecanone 1.00 2.00 1.14 2.24 3.43 1-Dodecanol, 3.7.11-trimethyl 0.08 0.17 0.05 0.12 0.31 2-Tridecanone 2.82 1.99 3.71 2.10 1.05 Epiglobulol 0.20 0.36 0.17 0.33 0.25 Elemol 1.10 0.77 0.50 0.81 0.30 2-Tetradecanone 0.13 0.50 0.32 0.15 0.22 Nepetalactol 0.47 0.31 0.28 0.97 0.65 Ascaridole 0.77 0.48 0.36 0.04 1.00 Guaiol 0.29 0.30 0.34 0.20 0.12 Eudesmol 0.13 0.22 0.41 0.11 0.03 Hexadecanal 0.14 0.18 0.09 0.03 0.12 9,12,15-Octadecatrienal 0.32 0.76 0.55 0.17 0.58 Hexadecanoic acid 0.45 0.82 0.23 0.16 0.09 3-Ethoxy-4-hydroxy-4-(40.11 0.21 0.10 -0.04
methoxyphenyl)cyclopent-2-enone
9,12,15-Octadecatrienoic acid methyl ester

Mean
2.12 1.02 2.43 12.64 1.25 0.08 0.51 20.05 0.21 12.46 0.91 0.82 0.64 53.33 1.96 0.15 2.33 0.26 0.70 0.26 0.54 0.53 0.25 0.18 0.11 0.48 0.35 0.09 1.64

1.10

1.96

1.10

0.93

3.11

Total oxygenated 76.64 80.82 79.54 69.46 84.59 78.21 Total identified 97.77 99.73 99.64 95.98 98.18 98.26 1 = Control 2 = Yeast + NPK 3 = EM + NPK 4 = Azotobacter + NPK 5 = Compost + NPK

Table (7): Effect of some fertilizer treatments on flowers essential oil constituents of Ruta graveolenus L. plants Compounds 1 2 3 4 5 Mean Hydrocarbon compound Limonene 1.88 3.24 5.91 1.65 2.29 2.99 Geyrene 0.96 0.49 1.23 2.50 1.83 1.40 1-Nonene 1.54 1.21 1.16 0.96 2.00 1.37 2-Nonene 1.97 2.29 1.84 1.22 2.65 1.99 1-Undecene 5.9 4.55 2.94 6.36 3.87 4.72 Anthracene 0.34 0.27 0.35 0.40 0.40 0.35 Neophytadiene 0.08 0.65 0.27 0.35 0.55 0.38 3,4-Dihydrobenzo[b]fluoranthene 0.16 1.92 1.02 0.80 1.74 1.13 Pentacosane 0.47 0.24 0.15 0.14 0.33 0.27 Total hydrocarbon 13.30 14.86 14.87 14.38 15.66 14.61 Oxygenated compound 2-Nonanone 11.3 12.26 8.26 6.91 8.02 9.35 Cyclotridecanone 0.19 0.08 0.31 0.17 0.22 0.19 2-Docanone 1.50 1.93 2.55 2.77 3.98 2.55 67.69 63.11 67.00 70.10 66.45 2-Undecanone 66.87 2-Dodecanone 2.95 4.02 2.99 2.18 3.10 3.05 2-Tridecanone 0.41 0.27 0.24 0.13 0.35 0.28 1-Dodecanol, 3,7,11-trimethyl 0.22 0.19 0.12 0.07 0.09 0.14 2-Tridecanone 0.55 0.69 0.84 0.86 0.85 0.76 Epiglobulol 0.07 0.22 0.15 0.07 0.08 0.12 Elemol 0.12 0.10 0.20 0.06 0.09 0.11 2-Tetradecanone 0.10 0.03 0.09 0.04 0.05 0.06 Cycloundecanone 0.09 0.03 0.12 0.06 0.06 0.07 Guaiol 0.08 0.11 0.29 0.05 0.08 0.12 Eudesmol 0.10 0.21 0.41 0.22 0.11 0.21 9,12,15-Octadecatrienal 0.08 0.11 0.07 0.09 0.12 0.09 Hexadecanoic acid 0.02 0.06 0.05 0.02 0.09 0.05 9,12,15-Octadecatrienoic acid methyl ester 0.92 1.00 0.99 0.81 0.07 0.76 Total oxygenated 86.39 84.42 84.68 84.61 83.81 84.78 Total identified 99.69 99.28 99.55 98.99 99.47 99.40 1 = Control 2 = Yeast + NPK 3 = EM + NPK 4 = Azotobacter + NPK 5 = Compost + NPK

Table (8): Effect of some fertilizer treatments on rutin and cumarin content in Ruta plant during two seasons 2005 / 2006 and 2006/2007. Characters Rutin Cumarin Leaves ml/plant Leaves ml/plant Treatments % % Control Yeast EM Azotobacter Compost NPK Yeast + ½ NPK EM + ½ NPK Azotobacter + ½NPK Compost + ½NPK LSD at 5% level Control Yeast EM Azotobacter Compost NPK Yeast + ½ NPK EM + ½ NPK Azotobacter + ½NPK Compost + ½NPK LSD at 5% level

2.02 2.08 2.11 2.10 2.10 2.10 2.08 2.13 2.10 2.12

First season 2005 / 2006 0.034 0.11 0.032 0.21 0.035 0.15 0.035 0.18 0.034 0.14 0.036 0.19 0.036 0.30 0.036 0.27 0.035 0.27 0.035 0.25

0.0018 0.0033 0.0026 0.0030 0.0023 0.0033 0.0052 0.0046 0.0046 0.0042

2.08 2.05 0.13 2.15 2.12 2.15 2.11 2.16 2.00 2.15

0.08 0.16 0.01 0.12 0.10 0.14 0.22 0.19 0.19 0.17

0.037 0.037 0.034 0.036 0.036 0.040 0.040 0.038 0.038 0.036

0.0014 0.0029 0.0018 0.0019 0.0017 0.0025 0.0043 0.0033 0.0036 0.0029

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