Bioactive constituents and allelopathic activities of the invasive weed Ranunculus sceleratus L . Nile Delta , Egypt

Allelopathy is the biochemical interactions between plants that results from the activity of different phytochemicals synthesized by higher plants. Many plants show pronounced allelopathic activity [1-4] due to their capability to synthesize variable allelochemicals that release into the environment by leaching from leaves, degradation of plant residues, volatilization and root exudation [5] and could influence the life of some surrounding plants and animals [6]. There are several classes of allelochemicals include phenolics, flavonoids, alkaloids, tannins, terpenoids and steroids [7]. Such allelochemicals influence plant growth and development and could be used to reduce weed pathogens and enhance crops yield [8].

INTRODUCTION study aimed to determine some secondary metabolites and to investigate their antioxidant and allelopathic activity.

Plant Material and Extraction
Shoot and root system of Ranunculus sceleratus L. were collected during vegetative stage in March from canal banks of drains, Nile Delta, Egypt. The species were identified according to Boulos [10]. The samples were air dried then ground into a fine powder using a grinder (IKA®MF 10-Basic Microfine-Grinder Drive, Breisgau, Germany) and stored in paper bags. Voucher specimen was kept in the herbarium of Botany Department, Faculty of Science, Damietta University.

DPPH Radical Scavenging Assay
The extracts radical scavenging activity was determined according to the reaction with a 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) then compared to the standard catechol. Antioxidant activity was determined as described by Lim and Quah [33] where two ml of 0.15 mM DPPH was added to 1 ml of the studied extracts in different concentrations (50 -400 mg ml -1 ). The solvent was used instead of the extract to prepare blank. The contents were incubated for 30 minutes in dark, the absorbance (A) was measured at 517 nm. The antioxidant activity was calculated as: % antioxidant scavenging activity = [1-(A sample/ A control )] x 100 IC 50 was calculated as the concentration of the antioxidants of the extract required to decrease initial concentration of DPPH radicals by 50%. The antioxidant activity of catechol was also assayed for comparison.

Allelopathic Assay
Chenopodium murale seeds were gathered from maize cultivated fields in the north delta coast in Gamasa city, Al-Dakahlia Governorate, Egypt. Seeds were sterilized by 0.3% calcium hypochlorite, rinsed by distilled water and dried again using filter papers at room temperature for 7 days [34].
Concentrations of 50, 100, 200, 300 and 400 mg ml -1 extracts were prepared using stock extract of 0.1 g/100ml. The osmotic concentrations were less than 0.1 Mpa that are not determining factor influencing germination [35]. The pH values were adjusted to 7 using 1M hydrochloric acid, then kept in refrigerator at 4 ºC for any further use [36].

Bioactive Metabolites
The concentration of the biologically active phytoconstituents in the shoot and root systems of R. sceleratus are presented in Table 1. The methanolic extract was used for determination of the R. sceleratus active ingredients. The concentration of total phenolics in root and shoot (27.54 and 15.33 mg/g dried weight) were higher than those of saponins (16.87 and 15.98 mg g -1 dry weight), tannins (12.06 and 8.63 mg/g dried weight), and flavonoids (9.96 and 6.87 mg/g dried weight). However, alkaloids expressed the lowest contents in roots and shoots (3.88 and 2.57 mg/g dried weight).

Antioxidant Activity
1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals were used for evaluation of the antioxidant scavenging activity of the methanolic extracts of the Ranunculus sceleratus by measuring the concentration of an antioxidant needed to decrease the initial DPPH concentration by 50% (IC 50 ). The IC 50 is inversely proportional to the antioxidant power where the lower the IC 50 , the higher the antioxidant activity. The evaluation of the antioxidant activity of the Ranunculus sceleratus extract is presented in Table 2. By increasing the plant extract concentration, their scavenging  activity increased. In case of shoot and root of R. sceleratus extracts the increase was up to 500 μg/ml where the scavenging activity was 54.92% and 57.50% respectively. Moreover, the IC 50 value of the R. sceleratus extract was 0.37 mg/ml and 0.34 mg/ml for shoot and root, respectively, compared to 0.15 mg/ml for catechol. The antioxidant potentiality of R. sceleratus was reported by many researchers [25,[37][38][39]. The antioxidant activity of R. sceleratus may be ascribed to the high content of phenolics [13], flavonoids [15], alkaloids [15], triterpene saponins [17] and essential oils [21]. The obtained results demonstrated that R. sceleratus antioxidant activity was higher than the results recorded by Neag et al. [39], but lower than those recorded by Shahid et al. [38].

Allelopathic Activity
The induced changes in germination and growth of seedlings under the influence of allelochemicals could be demonstrated using cell ultrastructure, molecular biology, in addition to biochemical and physiological characteristics [4,40]. The allelopathic potentiality of methanolic extracts from R. sceleratus (shoot and root) on germination and seedling growth of Chenopodium murale was evaluated using five different concentrations, and the results are presented in Figure 1. The inhibition was concentration dependent; meanwhile, root extracts more inhibition than shoot extract.
At 400 mg/ml, the germination of C. murale was inhibited by 79.74% and 92.64 for shoot and root extract, respectively, compared to control. However, the shoot growth was reduced by 76.06 % and 87.96 %, with the same sequence. The root growth was more sensitive to the allelopathic effect compared to the shoot, where it was inhibited by 82.68% and 98.67%, respectively, compared to control at the highest concentration ( Figure 1). The obtained data indicated that C. murale root growth was increasingly sensitive toward the allelopathic effect caused by R. sceleratus than shoot growth ( Figure 1) and can be attributed to the permeability of the root membrane as well as the direct touch with the allelochemicals [41][42][43]. Moreover, the roots exposed to the extract for longer periods and were the first to emerge [44].

CONCLUSION
In this study, the crude extract of Ranunculus sceleratus expressed more pronounced antioxidant capacity compared to the commercial antioxidant, which may be attributed to their high content of phenolics, tannins and saponins. However, Ranunculus sceleratus is considered as an invasive weed of orchards, roadsides and field crops [12], it may be used in controlling other weedy species through allelopathic application such as Chenopodium murale. These results support more studies to be carried for