The potential of Moringa oleifera extract as a biostimulant in enhancing the growth, biochemical and hormonal contents in rocket (Eruca vesicaria subsp. sativa) plants

Moringa oleifera is a highly valued plant, distributed in many countries of the tropics and subtropics. It has an impressive range of medicinal uses and high nutritional value. Accordingly, rocket (Eruca vesicaria subsp. sativa) plants were foliar sprayed with the aqueous extracts of leaves and twigs of M. oleifera at rates of 1, 2 and 3%. Among these concentrations, fertilization of rocket plants with 2% leaf and 3% twig extracts potentially increased all measured growth criteria (plant height, fresh and dry herb weight), photosynthetic rates, stomatal conductance, the amounts of each of chlorophyll a and b, carotenoids, total sugars, total protein, phenols, ascorbic acid, N, P, K, Ca, Mg, Fe as well as growth promoting hormones (auxins, gibberellins and cytokinins). Besides, bio-organic manuring with both kinds of Moringa extracts at all concentrations applied negatively reduced the levels of each of lipid peroxidation and abscisic acid as well as the activities of the antioxidant enzymes (catalase, peroxidase and superoxide dismutase). Thus, it is concluded that M. oleifera leaf and twig extracts can be recommended to be used effectively by farmers as a bio-organic fertilizer for various crops due to its high productivity, high nutritive value, antioxidant effect, easy preparation, low cost and environmentally friendly nature.


INTRODUCTION
Eruca vesicaria subsp. sativa (rocket or arugula) is an economically important leafy vegetable commonly found in the Mediterranean region, southern Europe and Central Asia (Pignone, 1997). Beside its culinary uses, rocket is also considered a medicinal plant, the extract of both plant and seed posses diversified therapeutic properties including antihyperlipidemic, antihyperglycemic, antinephrolithiatic, antiulcer, antiscorbutic, stimulant, stomachic and diuretic oil seed (Hungard et al., 1988;Pignone, 1997;Bukhsh et al., 2007;Alqasoumi et al., 2009). In addition, rocket has shown great potential as a green manure for controlling pathogenic fungi and parasitic nematodes as it contains chemicals with high biocidal activity that mimic synthetic fumigants (Ekaterini et al., 2006).
The dependency on the use of inorganic fertilizers as a source of plant nutrients by farmers and their high cost is further associated with land and soil degradation and environmental pollution (Phiri, 2010). Thus, there is continuous need to search for alternative safe natural sources of plant nutrients. Moringa oleifera is one of such alternative, being investigated to ascertain its effect on growth and yield of crops and thus can be promoted among farmers as a possible supplement or substitute to inorganic fertilizers (Phiri, 2010). Moreover, several researches have indicated that M. oleifera Lam (family: Moringaceae) is a highly valued plant with multipurpose effects (Yang et al., 2006;Anwar et al., 2007;Adebayo et al., 2011;Moyo et al., 2011;Mishra et al., 2011). The tree ranges in height from 5 to 10 m (Morton, 1991). It is found wild and cultivated in many countries of the tropics and subtropics (Morton, 1991). It is considered as one of the world's most useful trees, as almost every part of the tree has an impressive effect of food, medication and industrial purposes (Khalafalla et al., 2010;Adebayo et al., 2011;Moyo et al., 2011). Different parts of this plant contain a profile of important minerals, proteins, vitamins, β carotene, amino acids and various phenolics and provide a rich and rare combination of zeatin with several flavonoid pigments (Nagar et al., 1982;Siddhuraju and Becker, 2003;Anwar et al., 2007). So it is a good source of natural antioxidants (Anwar et al., 2007;Jacob and Shenbagaraman, 2011). Moringa seeds can be eaten fresh or cooked, or it can be pressed into sweet, nondessicating oil of high quality (30 to 40% of seed weight), commercially known as "Ben oil". Moreover, its unique property is the ability of the dry crushed seed and seed press cake which contain certain polypeptides to serve as a natural coagulants with antibacterial and antifungal activities, thus it has a compelling water purifying power (The Wealth of India, 1962;Ndabigengesere and Narasiah, 1998;Anwar et al., 2007). Concerning its medicinal value, it acts as cardiac and circulatory stimulants, posses antitumor, antipyretic, antiepileptic, anti-inflammatory, antiulcer, antispasmodic, diuretic, antihypertensive, cholesterol lowering, antioxidant, antidiabetic, hepatoprotective, antibacterial and antifungal activities and are being employed for the treatment of different ailments in the indigenous system of medicine particularly in South Asia (The Wealth of India, 1962;Morimitsu et al., 2000;Siddhuraju and Becker, 2003;Anwar et al., 2007;Moyo et al., 2011;Mishra et al., 2011;Jacob and Shenbagaraman, 2011).
Besides medicinal values of this plant , there has been earlier reports by, Fuglie (2000) that the leaf extract of M. oleifera accelerated growth of young plants, strengthened plants, improved resistance to pests and diseases, increased leaf duration, increased number of roots, produced more and larger fruits and generally increased yield by 20 and 35%. Several recent investigations were undertaken aiming to increase both the growth parameters measured as plant height, number of leaves, leaf area, fresh and dry weight, number of tillers, shoot vigor, root length, germination percentage and yield represented as fruit number and dry weight by foliar application of Moringa leaf extracts at different rates (Prabhu et al., 2010;Abdalla 43 Balakumbahan and Rajamani, 2010;Phiri, 2010;Nouman et al., 2011). Although various parts of M. oleifera plant extracts are known to possess diverse medicinal and biological activity on human and animals, little is known scientifically about its effect as a bio-organic fertilizer on the hormonal, metabolic and antioxidant potential on plants. Therefore, the present study was planned to explore the comparative and dose-dependent effect of the aqueous extracts of M. oleifera leaf and twigs in sustaining the growth, metabolic, hormonal and antioxidant activities of a leafy vegetable plant, rocket (Eruca vesicaria subsp. sativa).

Plant material and treatments
The seeds of rocket (E. vesicaria subsp. sativa c.v. Balady) were obtained from the Agricultural Research Center, Giza, Egypt. They were planted in plastic pots (20 cm in diameter) filled with equal amounts of clay loamy soil under controlled greenhouse conditions (relative humidity 35 to 50 ± 2%, average day/night temperature (25/18 ± 2°C) and irrigation was done following the common agricultural practice. The pots were arranged into 7 sets, 20 pots/set. The first set remained untreated to serve as control while the other 6 sets were fertilized with the biostimulant at different rates as follows: 0 (untreated control); 1L, 2L, 3L, 1T, 2T, 3T (M. oleifera leaf and twig extracts, each at 1, 2 and 3% respectively). After one week of planting and before foliar treatment, thinning was done so as to leave 10 plants/pot. The aqueous extract of M. oleifera tender leaves and twigs were prepared at the rates of 1, 2 and 3% using distilled water. The chemical analysis of both extracts was investigated using Fuglie (2000) and Moyo et al. (2011) and represented in Table 1. Foliar spraying of both extracts (leaves and twigs) were done twice at 7 and 14 days after planting (DAP). The plants were harvested at 50 DAP. Ten plants were randomly selected for the measurement of growth criteria and gas exchange rates. Other samples were taken (5 replicates/treatment) and were either oven dried for determinations of carbohydrates, nitrogen, phenols, ascorbic acid, total proteins and minerals or rapidly frozen for the estimations of antioxidant enzyme activities, photosynthetic pigments and phytohormones.

Measurement of gas exchange rates
Photosynthetic rate and stomatal conductance were measured using an open gas portable photosynthesis system (LI6400, LICOR, BioSciences, USA). Measurements were performed on sunny days under light conditions and between 9.00 and 12.00h on the upper most fully expanded leaves of 10 plants randomly chosen per treatment and expressed on a leaf area basis (Renault et al., 2001).

Chemical analysis
Photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) were determined spectrophotometrically (Metzner et al., 1965). Carbohydrate fractions were extracted, clarified and determined as total sugars (TS) (Dubois et al., 1965). Total proteins were estimated by Lowry et al. (1951) method. Ascorbic acid, a scavenger of oxyradicals was assayed by the method described by Roe and Keuther (1953) where ascorbate is converted to dehydroascorbate by the treatment with activated charcoal. Dehydroascorbic acid then reacts with 2,4-dinitrophenyl hydrazine to form osazones, which dissolves in sulphuric acid to give an orange colored solution, whose absorbance can be measured spectrophotometrically at 540 nm. Total phenols were determined in the ethanolic extract following the method described by Simons and Ross (1971) using folin reagent.
For the assay of antioxidant enzymes (catalase, CAT; peroxidase, POD; superoxide dismutase, SOD), fresh material were extracted following the method of Guerrier and Strullu (1990). The activities of CAT and POD were determined according to Chance and Maehly (1955). CAT activity was determined by measuring the decomposition of H2O2, by following the decline in its absorbance at 240 nm for 3min. POD activity was assayed by measuring the oxidation of guaiacol and the increase in absorbance at 470 nm was recorded in 3 min. The activity was defined as OD/min/mg FW. SOD activity was assayed by the nitrobluetetrazolium (NBT) modified method from that described by Dhindsa et al. (1980). One unit of SOD was defined as that being contained in the volume of extract that caused a 50% inhibition of the SOD-inhibitable fraction of the NBT reduction. The level of lipid peroxidation in tissues was measured by the determination of nmole of malonodialdehyde (MDA) formed using an extraction coefficient of 155 nmole L -1 cm -1 . MDA was determined using 20% trichloroacetic acid containing 0.5% thiobarbituric acid (TBA) reaction and the developed color was extracted with 2 ml n-butanol and the absorbance was measured at 530 nm. The value for the non-specific absorption at 600 nm was subtracted (Zhao et al., 1994).
Mineral elements were extracted from tissues similar to that of Chapman and Pratt (1961). Phosphorus was determined following the method described by Humphries (1956). Potassium was estimated photometrically according to Williams and Twine (1960). Calcium, magnesium and iron were determined by atomic absorption spectrophotometer according to AOAC, (1984). Total-N was determined by micro-Kjeldahl, Tector model 1026 after digestion in sulphuric acid (Horwilz, 2002).

Hormonal analysis
Growth regulators were estimated by collecting fresh samples in cold redistilled 95% ethanol in glass stoppered brown jars and kept in a deep freeze ready for the further analysis process. The method of extraction was essentially adopted by Wasfy et al. (1974). The fraction of the ethanol extract was carried out according to the method described by Shindy and Smith (1975). The acidic fraction contain the acidic hormones (IAA, GA3 and ABA) while the aqueous fraction comprised the cytokinins. The growth promoters (auxins, gibberellins, cytokinins) and the growth inhibitors (abscisic acid) were quantified using high performance liquid chromatography (HPLC) according to the method adopted by Muller and Hilgenberg (1986).

Statistical analysis
Morphologic and gas exchange values (photosynthetic rate and stomatal conductance) are means ± standard errors of 10 replicates while those of chemical and hormonal analysis are means ± standard error of 5 replicates. Significant differences were calculated using student's (t) test. SPSS version 15 was performed for multiple comparisons.

Growth parameters
Results presented in Table 2 show that M. oleifera leaf and twig extracts at 2 and 3%, respectively significantly enhanced the height of rocket plants as compared to untreated and differently treated plants. Also, the highest percentage increase in fresh herb weight of 68.1 and 61.7% per plant was recorded from the treatments 2L (2% leaf extract) and 3T (3% twig extract), respectively, while the lowest fresh herb weight was obtained in the untreated control plants. Similarly, the maximum percentage increase in dry herbage weight of 51.5 and 42.6% was recorded from the same treatments 2L and 3T and were significantly different from the other treatments and control. It was thus apparent that these two treatments (2L and 3T) among the other five treatments caused maximization of yield which is the ultimate goal to be achieved in any crop management aspect. Consistent results were obtained by Prabhu et al. (2010) and Balakumbahan and Rajamani (2010) by applying Moringa leaf extract at 2 and 4% on sacred basil and senna plants, respectively. They found that foliar spraying of Moringa leaf extract at 2% was more effective than 4% and raised all measured growth parameters above control plants (plant height, number of leaves, leaf area, leaf area index, fresh and dry weight, number of pods, number of branches, dry leaf yield and dry pod yield). The possible reason for this acceleration of growth might be due to the enriched content of Moringa leaf and twig extracts of crude proteins (43.5%) and growth promoting hormones, that is, auxins and cytokinins (Makkar and Becker, 1996;Moyo et al., 2011). Proteins are essential for the formation of the protoplasm, while growth hormones favored rapid cell division, cell multiplication and enlargement.

Gas exchange measurements, photosynthetic pigments, carbohydrate and soluble protein contents
When rocket plants were treated with 2% Moringa leaf extract (2L) they manifested an optimum photosynthetic rate and stomatal conductance ( Karanatsidis and Berova (2009) using different organic fertilizers who attributed these increases in the photosynthetic ability in plants in response to organic fertilization due to various factors namely the efficiency of PS∏, the stability of chloroplast ultra structure, the enhanced rate of CO 2 absorption by leaf cell and its fixation.
It was prominent from Table 3 that application of Moringa leaf extract at the medium dose (2L) exhibited the highest concentrations of photosynthetic pigments, namely, chlorophyll a, chlorophyll b and carotenoids as compared to those of untreated control plants. In addition, spraying rocket plants with the twig extract at the three rates 1, 2 and 3% elicited gradual increases in the amounts of these photosynthetic pigments but these increases were comparably less than those of Moringa leaf extract. Corroborative data were obtained using different types of organic fertilizers as compost, vermincompost, animal manure and seaweeds (Amujoyegbe et al., 2007;Noori et al., 2010;Abdalla and El-Khoshiban, 2012). The present knowledge ascertained that Moringa (leaves, seeds, pods) contains appreciable amounts of specific plant pigments with demonstrated potent antioxidant properties such as the carotenoids (lutein, alphacarotene, beta-carotene and xanthin) and chlorophyll (Owusu, 2008). Besides that, the leaves have high nutritional potentialities of several macro elements as Mg (Yameogo et al., 2011), a constituent of chlorophyll, both would account for the increase in the amounts of chlorophyll a and chlorophyll b in Eruca plants.
Rocket plants treated with the four concentrations of Moringa leaf and twig extracts (1L, 2L, 2T and 3T) showed significant accumulations of total sugars and total proteins and the maximum increments of both were obtained when the plants were treated with 2% leaf extract (2L), while the other two concentrations (3L and 1T) manifested marginal increases in both total sugars and total proteins above those of untreated control plants (Table 3). Among the two Moringa extracts examined, the leaf extract was found superior than the twigs towards the values of both total sugars and total proteins in Eruca plants. Foidl et al. (2001) reported that foliar spraying of some plant leaves with Moringa extract produced some notable effects as overall increase in plant yield between20 and 35% and higher sugar and mineral levels. The enhanced accumulations of both total protein and total sugars in rocket plants in response to treatments with Moringa leaf and twig extracts were due to the high protein, sugar and starch content of the entire M. oleifera plant (Table 1) and which make it of great scientific and agricultural interest (Foidl et al., 2001;Yameogo et al., 2011).

Antioxidant compounds and antioxidant enzyme activities:
With increasing rates of Moringa leaf and twig extracts, either progressive declines in each of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activities and the levels of lipid peroxidation (measured as nmole of MDA formed) or gradual increases in phenol and ascorbic acid (AA) amounts were observed so as to reach minimum and maximum increments at 2L and 3T, respectively, as compared to those of untreated plants (Table 4). Data in Table 4 clearly showed that Eruca plants sprayed with Moringa twig extract exerted higher antioxidant enzyme activities (SOD, POD and CAT) and MDA level and lower antioxidant compound levels (phenols and AA) than those treated with the leaf extract which reflects the booster effect of the leaf extract as an antioxidant.
Total phenols comprise the largest group of plants secondary metabolite. They can directly react with superoxide anions and lipid peroxyl radical and consequently inhibit or break the chain of lipid peroxidation (Rajanandh and Kavitha, 2010). Ascorbic acid (AA) is a familiar molecule because of its dietary significance, it is not only an important antioxidant, it also appears to link flowering time, developmental senescence, programmed cell death and responses to pathogens through a complex signal transduction network (Nicholas, 1978;Talreja, 2011). They induces better root and shoot growth, maintain higher leaf moisture content and lower disease incidence in both normal and stressful environments (Zhang and Schmidt, 1999).
Under normal circumstances, the reactive oxygen molecules (ROS) such as O 2 -,H 2 O 2 and OH are continuously generated and detoxified by the antioxidant compounds (phenols, flavonoids, carotene and AA) and antioxidant enzymes (SOD, POD and CAT) present in the tissues.
Thus, there is equilibrium between the ROS generated and the antioxidant present. However, owing to ROS over production and/or inadequate antioxidant defense, this equilibrium is hampered favoring the ROS upsurge that culminates in oxidative damage to various biomolecules including proteins, lipids, lipoproteins and DNA (Sreelatha and Padma, 2009).
M. oleifera aqueous extract possessed a wide spectrum of dietary antioxidant as phenols, flavonoids, βcarotene, AA, α-tochopherol (vitamin E) and antioxidant enzymes (SOD, POD and CAT). Thus, the extract exhibited strong scavenging effect on 2,2-diphenyl-2-picryl hydrazyl (DPPH) free radical, superoxide, nitric oxide radical and inhibition of lipid peroxidation as compared to many reference antioxidant extracts of various leafy vegetables and fruits (Dasgupta and De, 2006;Sreelatha and Padma, 2009;Jacob and Shenbagaraman, 2011). Thus, this extract can prevent oxidative damage to major biomolecules and afford significant protection against oxidative damage by favoring the accumulation of higher levels of AA and phenols which, in turn, rendered the activities of antioxidant enzymes and lipid peroxidaton in rocket plants under investigation to minimum levels.

Inorganic nutrient contents
It is evident from Table 5 that there were positive correlations between the increased rates of both extracts of Moringa applied up to 3% and the accumulation of both major (N, P, K, Ca and Mg) and minor (Fe) elements estimated, in the present study, in rocket plants. The uptake and accumulation of these nutrients in rocket plants were significantly higher due to fertilization with the leaf extract than the twigs. Several comparable studies confirmed the current data. For instance, Schuphan (2005), Noori et al. (2010), Sivakumar and Ponnusami (2011) and Abdalla and El-Khoshiban, (2012) realized the increased uptake and accumulations of some nutritive elements as N, K, Ca, Mg, P as well as Fe in roots and shoots of several plants under investigation as a consequence of organic fertilization from different sources (plant and animal  source) including Moringa leaf extract. Sivakumar and Ponnusami (2011) indicated that organic manures are fairly good source of nutrients which boosted plants to uptake progressively beneficial elements, to increase the leaf nutrient status and eventually attain optimum growth and productivity. Bio-organic fertilization is supposed to accelerate the nutrient uptake through the tested rocket plant by increasing the permeability of root membranes for electrolytes, preventing their fixation in the soil and increasing their mobility. Different part of M. oleifera plants have been reported to be a rich source of important minerals as Ca, Mg, K, Fe, Zn, P, S, Cu, Mn , Se and Na which can be valorized for a balanced nutrition of populations (Yameogo et al., 2011 ;Moyo et al.,2011).

Hormonal analysis
The changes in the phytohormonal levels of untreated and biofertilized rocket plants are presented in Table 6. The contents of growth promoting hormones (auxins, gibberellins and cytokinins) increased progressively above those of the corresponding controls in a dose related manner so as to reach maximum levels either at 2L or 3T in response to fertilization with Moringa leaf and twig extracts respectively. Reversibly, supplementing rocket plants with both leaf and twig extracts negatively reduced the abscisic acid (ABA) values either markedly (1L, 2L, 2T, 3T) or marginal (3L, 1T) below those of untreated plants (Table 6). Noteworthy, in the current work, the above results of the different values of phytohormones accommodated well with those of growth criteria due to foliar spraying of rocket plants with Moringa leaf and twig extracts at three rates: 1, 2 and 3%. Consistent results were obtained by Makkar and Becker (1996) and Anwar et al. (2007). They isolated growth promoting hormones (zeatin and auxins) from Moringa leaf extract and used this extract as a foliar spray to accelerate growth of different plants. Results showed that foliar treatment with Moringa extract increased flowering, dry matter, fruit weight, produced larger flowers and fruits and consequently higher yield at harvest time, greater number of shoots per plant and higher percentage of sugars and minerals and eventually caused plants to be firmer and more resistant to pests and diseases (Foidl et al., 2001).

Conclusion
Overall, based on the current results, Moringa leaf and twig extracts can be used at rates of 2 and 3%, respectively to stimulate the biomass production of Eruca plants, enhanced photosynthetic pigments, total sugars, total proteins, growth promoting hormones (auxins, gibberellins and cytokinins) and various essential mineral elements (N, K, Ca, Mg, P and Fe). In addition, the present results support the potent antioxidant activity of Moringa extracts as it successfully increased the phenol and AA contents, whereas decreased the activity of each of SOD, CAT and POD and lipid peroxidation levels in rocket plants, which adds one more positive attribute to its known pharmacological properties and hence its use in traditional system in medicine.
Furthermore, the presence of high levels of inorganic and organic matter in the extracts of this plant, it's easy preparation and its ecofriendly nature suggest the greater practical importance of Moringa extracts. Thus, with the current thrust on sustainable agriculture and organic farming, the use of M. oleifera extract should grow in popularity and led to development of a large number of Moringa extract products that can be employed either as root tips, soil drenches or foliar sprays. Subsequently, it is concluded that this tree has the potential to improve nutrition, boost food security and foster rural development.

ACKNOWLEDGMENTS
The author is thankful to the Biotechnology Department, National Research Center, Dokki, Giza, for performing mineral analysis by atomic absorption spectrophotometer and hormonal analysis by high performance liquid chromatography (HPLC). Sincere thanks and obligations are also extended to Dr. Raifa A. Hassanein, Prof of Plant Physiology, Faculty of Science, Ain Shams University, for her valuable advice and to my husband who suggested the idea and encouraged me throughout this work.