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, B-
vitamins 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 (%) Silt (%) Clay (%) pH
Loamy 8.7 30.0 29 30.0 7.8
EC (mmhos/cm) N P K S Na Ca Mg Fe Zn Mn Cu
6.5
(ppm)
14.0 0.54 3.0 4.2 8.0 9.2 14 3.0 4.5 2.0 0.80

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 pH EC Total Organic Ash C/N P K Fe Mn Cu Zn
% (N%) matter % ratio % % ppm ppm ppm ppm
%
27-46 7.2-7.6 3-4 1.4-1.7 38-50 41-43 13.6:1 0.47 1.2-1.6 800 190 40 70
7
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 (Ben-
Hammouda 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
(cm) g/plant Kg/fed. g/plant Kg/fed.
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 g/plant Kg / fed. g/plant Kg / fed.

First season 2005 / 2006
Control 5.23 78.45 0.68 10.20
Yeast 10.33 154.95 1.34 20.14
EM 7.29 109.20 0.95 14.20
Azotobacter 8.49 127.35 1.10 16.56
Compost 6.67 100.05 0.87 13.01
NPK 9.14 137.10 1.19 17.82
Yeast + ½ NPK 14.33 214.95 1.86 27.94
EM + ½ NPK 12.64 189.60 1.64 24.65
Azotobacter + ½NPK 13.00 195.00 1.69 25.35
Compost + ½NPK 11.98 179.70 1.58 23.36
LSD at 5% level 1.18 12.10 0.23 1.30
Second season 2006 / 2007
Control 3.86 58.05 0.50 7.50
Yeast 7.76 116.40 1.01 13.13
EM 5.17 77.54 0.67 10.08
Azotobacter 5.39 80.86 0.70 9.10
Compost 4.75 71.25 0.62 9.30
NPK 6.28 94.20 0.82 12.25
Yeast + ½ NPK 10.66 159.90 1.39 20.85
EM + ½ NPK 8.75 131.25 1.14 17.10
Azotobacter + ½NPK 9.35 140.25 1.22 18.30
Compost + ½NPK 8.13 121.95 1.06 15.90
LSD at 5% level 2.50 10.52 0.30 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 El-
Sherbeny (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
Pyrogallic acid 112.13 80.23 101.24 130.10 145.0 33.18 66.97 44.63 82.21 25.90
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
Salicylic acid 176.22 155.27 236.14 223.14 166.27 118.01 93.65 175.30 100.35 92.83
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
Cinnamic acid 12.41 24.10 18.41 30.12 27.20 12.12 8.51 25.90 11.65 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
Herb

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
Flowers

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)
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 28-
33 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 hydroxy-
4-(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-(4-
methoxy-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 Mean
Hydrocarbon compound
Limonene 2.00 2.25 4.12 1.22 1.03 2.12
Geyrene 1.17 0.81 1.05 1.05 1.00 1.02
Nonene 1.90 2.55 3.52 3.32 0.88 2.43
Undecene 14.00 10.15 9.60 19.76 9.68 12.64
Anthracene 1.62 2.00 1.00 0.88 0.75 1.25
Neophytadiene 0.11 0.03 0.03 0.18 0.05 0.08
3,4-Dihydrobenzo[b]fluoranthene 0.33 1.12 0.78 0.11 0.20 0.51
Total hydrocarbon 21.13 18.91 20.10 26.52 13.59 20.05
Oxygenated compound
2-Octanone 0.30 0.26 0.11 0.27 0.12 0.21
2-Nonanone 20.00 16.11 10.60 6.37 13.23 12.46
Teteradecanal 0.18 1.00 0.51 0.92 1.96 0.91
Dodecanal 0.95 1.00 1.19 0.40 0.58 0.82
2-Docanone 0.66 0.27 1.88 0.09 0.31 0.64
2-Undecanone 49.44 51.15 55.90 53.05 57.09 53.33
2-dodecanone 1.00 2.00 1.14 2.24 3.43 1.96
1-Dodecanol, 3.7.11-trimethyl 0.08 0.17 0.05 0.12 0.31 0.15
2-Tridecanone 2.82 1.99 3.71 2.10 1.05 2.33
Epiglobulol 0.20 0.36 0.17 0.33 0.25 0.26
Elemol 1.10 0.77 0.50 0.81 0.30 0.70
2-Tetradecanone 0.13 0.50 0.32 0.15 0.22 0.26
Nepetalactol 0.47 0.31 0.28 0.97 0.65 0.54
Ascaridole 0.77 0.48 0.36 0.04 1.00 0.53
Guaiol 0.29 0.30 0.34 0.20 0.12 0.25
Eudesmol 0.13 0.22 0.41 0.11 0.03 0.18
Hexadecanal 0.14 0.18 0.09 0.03 0.12 0.11
9,12,15-Octadecatrienal 0.32 0.76 0.55 0.17 0.58 0.48
Hexadecanoic acid 0.45 0.82 0.23 0.16 0.09 0.35
3-Ethoxy-4-hydroxy-4-(4- 0.11 0.21 0.10 -- 0.04 0.09
methoxyphenyl)cyclopent-2-enone
9,12,15-Octadecatrienoic acid methyl ester 1.10 1.96 1.10 0.93 3.11 1.64
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
2-Undecanone 67.69 63.11 67.00 70.10 66.45 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 % %
First season 2005 / 2006
Control 2.02 0.11 0.034 0.0018
Yeast 2.08 0.21 0.032 0.0033
EM 2.11 0.15 0.035 0.0026
Azotobacter 2.10 0.18 0.035 0.0030
Compost 2.10 0.14 0.034 0.0023
NPK 2.10 0.19 0.036 0.0033
Yeast + ½ NPK 2.08 0.30 0.036 0.0052
EM + ½ NPK 2.13 0.27 0.036 0.0046
Azotobacter + ½NPK 2.10 0.27 0.035 0.0046
Compost + ½NPK 2.12 0.25 0.035 0.0042
LSD at 5% level

Control 2.08 0.08 0.037 0.0014

Yeast 2.05 0.16 0.037 0.0029
EM 0.13 0.01 0.034 0.0018
Azotobacter 2.15 0.12 0.036 0.0019
Compost 2.12 0.10 0.036 0.0017
NPK 2.15 0.14 0.040 0.0025
Yeast + ½ NPK 2.11 0.22 0.040 0.0043
EM + ½ NPK 2.16 0.19 0.038 0.0033
Azotobacter + ½NPK 2.00 0.19 0.038 0.0036
Compost + ½NPK 2.15 0.17 0.036 0.0029
LSD at 5% level
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