Bioactive Compounds and Biological Assays of different Prosopis Juliflora Extracts against Groundnut Aphid

The high diversity of plants is an available source of useful compounds. People extract plants and use them for different purposes. Ancient habitats found plant extracts as effi cient medicines for the relief of pains or alleviation of the symptoms of the disease, as poisons for use in warfare and hunting, as effective agents for euthanasia and capital punishment. They were also used as narcotics, hallucinogens, or stimulants to relieve the tedium, or alleviate fatigue and hunger. Many of these natural products are still used today and usually for the same general purposes [1].


Introduction
The high diversity of plants is an available source of useful compounds. People extract plants and use them for different purposes. Ancient habitats found plant extracts as effi cient medicines for the relief of pains or alleviation of the symptoms of the disease, as poisons for use in warfare and hunting, as effective agents for euthanasia and capital punishment. They were also used as narcotics, hallucinogens, or stimulants to relieve the tedium, or alleviate fatigue and hunger. Many of these natural products are still used today and usually for the same general purposes [1].
Natural products are organic compounds of natural origin that are unique to one organism or common to a small number of closely related organisms. The use of natural products as medicines, poisons, hallucinogens, stimulants, perfumes, fl avoring agents, insecticides, insect antifeedants, fungicides, plant growth-regulating hormones, molluscicides, etc., is well known. It is therefore not diffi cult to understand what motivates chemists to isolate and characterize natural products. The characterization of new compounds is usually followed by their synthesis and the study of their biological activity and biosynthesis. One of the most interesting phenomena that emerged in the last few years is the realization that natural products that have been considered useless do have functions in the organisms from which they originate. It is recognized that many of them have vital roles as mediators of ecological interactions, thereby ensuring the continued survival of a particular organism. Despite the vast number and structural diversity of metabolites, almost all arise by one of three biosynthetic pathways or by a combination of two or more of these pathways. These are known as the acetate, mevalonate and shikimate pathways [2]. are native to the Americas, three to Asia and one to Africa.
In the Americas, Argentina has 28 native species, of which 13 are endemic [3]. Prosopis species grow in a wide array of environments and are commonly not restricted by soil type, pH or salinity. They grow in semi-arid and arid tracts of tropical and sub-tropical regions of the world and are spreading fast because the leaves are unpalatable and animals do not digest its seeds [4]. P.julifl ora is a shrub or small tree, a kind of mesquite.
Within Prosopis species, there are trees and shrubs of varying size, mainly characterized by the presence of thorns and prickles. The term "mesquite" includes all leguminous trees of the genus Prosopis, of which nine species are found in the highly arid environments of Mexico. These species are highly recognized for their properties as windbreakers, soil binders, and sand stabilizers, as well as their ability to grow in the poorest soils and to survive in areas where other trees cannot survive [5]. P.julifl ora was introduced in Ethiopia as a biological soil and water conservation agent during the late 1970s. Now it is becoming a major threat because of its invasive nature.
Some reports indicate that P. julifl ora is widely distributed in Ethiopia [6,7].
Leaves and extracts of the leaves of Prosopisspecies have been noted to have bio-control properties. Extracts of P. julifl ora are effective against some weed species, insects, nematodes, pathogenic fungi and viruses. Leaf extracts from P. julifl ora have been found to inhibit germination in a number of species ) [8]. The uses of P. julifl ora leaves to control the invasive weed Parthenium hysterophorus has been assessed, with a noted reduction in seed germination [9].
The numerous goods and services obtained from P. julifl ora have led to global introductions of the species. In South Africa, pods are collected to produce organic medicines ('manna') that are said to have properties that stabilize blood sugar level in humans [10]. Products from this plant have also been used for human consumption in bread, biscuits, sweeties, syrup and liquors [11]. Extracts of P.julifl ora seeds and leaves have shown several in vitro pharmacological effects such as antibacterial, antifungal, and anti-infl ammatory properties [12]. The aqueous extracts of P. julifl ora have also been known for its antibacterial activity against different phytopathogenic bacteria [13]. A water-soluble mixture of alkaloids from P. julifl ora leaves was found to be more active against gram-positive bacteria than commercial antibiotics such as bacitracin, chloromycetin, gentamicin or trimethoprim [14]. Therefore, the objective of this study was to investigate the phytochemical composition of P.julifl ora and insecticidal activities of its extracts against groundnut aphid.

General
All solvents, standards and reagents used were of the analytical grade, that purchased from Fisher Scientifi c UK, Bishop Meadow Road Loughborough, Leics, LEll 5RG, UK, except for fatty acid standards that were purchased from Sigma-Aldrich Chemie GmbH, Riedstrasse2, D-89555 Steinheim Germany.

Instruments
GCMS analysis was carried out with Agilent technologies 7820A GC and 5977E MSD system equipped with an autosampler. Chromatographic separations were carried out using column length 30 m × 0.25 mm and column phase thickness 0.25 μm of HP-88 coated with 100% poly (dimethylsiloxane). Injection mode was splitless and 1μl injection volume in total run time 31.31 minutes under helium carrier gas was injected. The fatty acid methyl esters were run on GCMS and their results were expressed as mean values ± standard deviation (M±STD).
NMR spectra were recorded on Bruker Avance 400 MHz spectrometers. For 1 H NMR (400 MHz) and 13 C NMR (100.6 MHz) spectra, the chemical shifts () are reported in parts per million (ppm) relative to TMS. The  values are referenced to CHCl 3 in CDCl 3 at 7.27 ppm.

Chromatography
Analytical TLC was run on MerckKieselgel 60 F254. Plates were visualized under UV light and by spraying with Vanillin:5% H 2 SO 4 in MeOH followed by heating for a few seconds.

Plant material
The stem bark, seeds and leaves of P. julifl ora were collected from Amibara Woreda (Afar region) 9 o 60' 45" N latitude and 40 o 9' 32" E longitude and at an altitude of 740 meters above sea level), 280 km northeast of Addis Ababa, during October 2016. The plant specimen was identifi ed at the Biology Department, AAU. The samples were collected in sterile polyethylene bags. The fresh samples were transported in icebox and were preserved in a deep freezer until processing.

Plant materials preparation
The stem bark of P. julifl ora was chopped into small pieces and dried at room temperature for two weeks. The dried seeds and stem bark were milled using a "knife" mill. The fresh leaves were frozen in liquid nitrogen and crushed with a mortar and pestle.

Extraction
The powdered plant materials were extracted by hydrodistillation, Soxhlet, and solvent extractions. The crude extracts were evaluated for the insecticidal activity against groundnut aphids under laboratory conditions with different concentrations. Based on the preliminary insecticidal evaluation phytochemical study was conducted on methanol leaf extract of P. julifl ora as follows.
The fresh leaf of P. julifl ora was cut into small pieces, frozen in liquid nitrogen and ground. The ground leaf (5 kg) Citation: Zerihun  fi lter paper. The solvent was removed by a rotary evaporator at 35 °C to afford a dark green crude extract (309 g). Solventsolvent partition (Figure 1) was done by using the protocol developed by Kupchan and Tsou [15] and modifi ed by Wagenen, et al. [16]. Thus, the crude extract was dissolved in MeOH  Figure   1 summarizes the extraction procedure.

Isolation of compounds
The residue (FR 3, 30g) was applied on a silica gel column and eluted with DCM-MeOH mixtures of increasing polarity. A total of 49 fractions (each 100 mL) were collected as shown in Table 1. The progress of separation was monitored by analytical TLC with DCM-MeOH solvent system and fractions of similar TLC profi les were combined to give twelve fractions (Table 1). Fraction 6' was subjected to further purifi cation over a short silica gel column with EtOAc:MeOH mixtures in increasing polarity. A total of 13 fractions (each 10 mL) were collected. Fractions 1 to 5 were collected using EtOAc-methanol (9:1) solvent system. Fraction 6 eluted with EtOAc:MeOH

Identifi cation of compounds
The structures of isolated compounds were elucidated using physical parameters and spectroscopic techniques. Detailed analysis is given in the results and discussion sections.

Bioassays
The insecticidal activities of stem bark, seed and leaves extracts of P. julifl ora were evaluated using dosage-dependent were used in these bioassays. In the case of essential oil, an equivalent amount of deionized water and saturated sodium chloride were used as positive and solvent controls respectively.
In each treatment three replication, each containing 10 aphids were used. Bioassays were carried out at room temperature (28 ± 2 °C) in 30 cm diameter petri dish plates containing circular 33 mm 2 Whatman #1 fi lter paper inside each well. 1mL sample solution was added to the upper part of the petri dish. After the application of the solutions, aphids were maintained at controlled temperature (28 ± 2 °C) and humidity (70% ± 10%).
Mortality rates were recorded after 12, 24, 48, 72 and 96 hrs after applying the solutions. Following the same procedure, dichloromethane and methanol extracts of seed, stem bark and leaves of P. julifl ora were evaluated for their insecticidal activities.  [17]. The present work has investigated the effects of extracts of P. julifl ora against groundnut aphid.

Results and discussion
The results obtained from this study are discussed below.

Bioassays of extracts of P. julifl ora against groundnut aphid
The insecticidal activities of extracts of P. julifl ora that were obtained through hydro distillation, solvent extraction and Soxhlet extraction were evaluated against groundnut aphid (Aphis craccivora). Insect mortality was evaluated using dosage-dependent bioassays as described in the experimental section and the results were recorded after 12, 24, 48, 72 and 96 h. All measurements were done in triplicate analysis and the results were recorded as mean ± standard deviation (SD).
Statistical analyses were indicated that all of the extracts showed a dependence between mortality and concentration (P < 0.0001) ( Table 2). The calculated mean mortalities of groundnut aphids are presented in Table 2.
The results presented in Table 2 summarize the bioassays of the extracts of P. julifl ora that have promoted statistically signifi cant mortality using Duncan's signifi cance test [18] for at least one concentration compared with the control.
Insecticidal activities higher than 50% at any tested concentrations were considered to show signifi cant differences.       toxic compounds which may act individually or synergistically against groundnut aphids (Table 3). Table 3. Effi cacy of P. julifl ora stem bark, seed and leaf extracts against groundnut aphids for lethal concentration LC 50 and LC 95 at the shortest time (12 h) and for lethal time LT 50 and LT 95 at the smallest concentration (1%) after treatments.

Phytochemical investigation of the leaves of P. julifl ora
In this work, attempts were made to isolate and characterize secondary metabolites from the leaves of P.
julifl ora. Thus, two compounds were isolated by using silica gel column chromatography and characterized through different spectroscopic techniques as described below.
The 1 H NMR spectrum of compound 1 (Table 4) showed the doublets at  7.1 and 6.79 (J = 8.0 Hz) integrating for two protons each due to aromatic protons on a 1,4-disubstituted benzene ring. Additionally, two triplets were observed at  3.12 and 2.87 (J = 8.0 Hz) integrating for two protons each indicating the presence of two adjacent methylene groups, presumably attached to the aromatic ring at one end and to an electronwithdrawing group at the other.
The 13 C NMR spectrum (Table 5) of compound 1 showed a total of six carbon resonances of which four were in the aromatic region and the remaining two were in the aliphatic region( Figure 3). The DEPT-135 spectrum showed that the signals at  129.4 and 115.3 were due to aromatic methine carbons while the signals at  40.9 and 32.4 were due to aliphatic methylene carbons. The quaternary carbon signals have appeared at  156.4, 127.0. The 13 C NMR spectrum together with the 1 H NMR spectrum allowed the assignment of the signal at  129.4 to the two equivalent aromatic carbons C-2 and C-6. The signal at  115.3 can be attributed to C-3 and C-6 ortho to an electron-donating group at C-4. It is thus evident that the side chain was attached to C-1 whose carbon resonance appeared at  127.0.
The UV, IR and NMR data were allowed for the identifi cation  HS= out of the range (highly signifi cant), M = mortality, y = slope, C = concentration b = y-intercept   4-(2-aminoethyl) phenol in the literature [21,22] revealed that the two compounds are identical( Figure 3).

Conclusion
The present study was conducted to evaluate the insecticidal activities of different extracts of P. julifl ora against groundnut aphids (Aphiscraccivora). All extracts of P. julifl ora showed a high percentage of mortality at 1% concentration in 24 h against groundnut aphids. Different extracts of P. julifl ora showed a signifi cant difference in the insecticidal activity at a 0.001% level of confi dence. Thus, P. julifl ora has the potential to be used as a bio-insecticide. As part of this research, the phytochemical Citation: Zerihun