Evaluation of synergistic medicinal efficacy of four alkaloidic compounds isolated from Iraqi Allium porrum seeds against staphylococcus aureus bacteria

Medicinal plants are necessary benefit natural sources for chemical production of different therapies from secondary metabolism. Allium porrum is considered as one of sources of foods and drugs at the same time. So the current work focused on isolation and characterization of four alkaloids from seeds of this plant. These compounds are 2-piperidinone, 2-pyrrolidinone-4-methyl, adenosine-2-deoxy–N-methyl –N-(triflouro acetyl ) and maymarsine by using Gas Chromatography – Mass Spectroscopy (GC-MS) technique. The medicinal activity was achieved to investigate and evaluate the synergistic action of the mixture of the four alkaloidic compounds towards Staphylococcus aureus pathogen. The concentrations of alkaloidic mixture were prepared with values equal to 50,100,125,150,200,250,300,375,400 and 500 mg/ml and they revealed inhibition zones with diameters represented by 31,31,32,35,35,37,41,43,43and 45 mm. So the mixture of the four alkaloidic compounds can be used as natural therapy to treat various inflammatory caused by this pathogenic bacteria.


Introduction
The social and healthy need to discover natural drugs more safe than synthetic antibiotic led to development and creation in science of medicinal plants . Pre studies about the natural chemical compounds insured and proved the medical value for the chemical components abundant in the different parts of plants so the active materials were isolated , characterized and carried out successfully in treat various diseasess caused by different microorganisms [1][2] [3] . The medical importance of all active metabolites is absence of the side effects causing to other infections and ,or inflammatory leading to a great disorder in human health. So the centers of  [4][5] [6] . Antibacterial activities were achieved for various medicinal plants containing a wide range of active chemical metabolites in the different parts of these plants . Also these studies insured the great potential of active secondary metabolites for inhibition the biological and biochemical functions of living systems belonging to the pathogenic microorganisms [7][8] [9] . Alluim porrum (leek) is one of medical plant belongs to Brassicaceae family . This plant is traditionally used as a food for human in the worldwide because of its nutrition value for the human health .There are 500 genus are available in various countries but they differ in some features such as colour, maturing time and taste .Also these botanic genera are similar in chemical composition and neutral ceuitical components . Different phytochemical studies were achieved about various parts of Allium porrum and they indicated and ensured xistence of active metabolites which included phenolics , active antioxidants sulphuric phenols and flavonoids [10] [11] .
Alkaloids are secondary active metabolites are found in most medicinal plants and these chemical compounds are anabolized in plants from the starting material is amino acid by different biochemical pathways by the support of required enzymes . Chemical alkalolidic metabolites are heterocyclic compounds containing nitrogen atom(s) and they have basic properties but some of them have weak acidic features whereas a few of them have neutral properties . In addition to presence of nitrogen atom(s) in the chemical structure contain sulpher , chloride , bromine and phosphorus atoms also are existing in various multi-alkaloids .Because alkaloidic metabolites are active compound so they have high medicinal efficacy to treat different diseases caused by multi-pathogens such as bacteria , virus and fungi [12][13] [14] .Therefore the following research was achieved to estimate and evaluate the medicinal efficacy of synergistic interaction of mixture of four alkaloidic compounds isolated and identified from Iraqi Allium porrum seeds against pathogenic bacteria represented by Staphylococcus eureus.

Preparation of plant
Leek (Allium porrum) seeds were purchared from the local market in Al-Ashar district at Basrah Governorate in Iraq ,then the plant was taxonomied in biology department -college of education for pure sciences at University of Basrah -Iraq by specialist botanist . The seeds were washed with distilled water , dried under the shadow ground as powder with weight equal to 100 gm by electrical mill and placed in dark containers until the day of use .

Preparation of the cold aqueous extract of Allium porrum
The aqueous extract belonging to seeds of Allium porrum was prepared by mixing of 50 gm of seeds with 500 ml of distilled water as solvent in a conical flask . The contents were stirred on magnetic stirrer for 10 hours at room temperature , then the mixture was filtered by using Buchner funnel . The precipitate was removed and the filterate was dried carefully to get a solid crude [15] with weigh equal to 6.25 gm .

Qualitative detections of alkaloidic compound
The cold aqueous extract of Allium porrum seeds was underwent Dragendroff reagent to detect the alkaloids existing in the plant . Fifty mg of extract was dissolved in 1 ml of distilled water in a test tube , then 2 ml of Dragendroff reagent was added into tube then orange precipitate was fromed [16] .

Isolation of alkaloids from Allium porrum
Fifty grams from seeds powder of Allium porrum was added into 500 ml of ethanolic acetic acid has concentration equal to (10% v/v) in conical flask and the contents were mixed and then stirred by using magnetic stirrer for 12 hours . The mixture was filtered by Buchner funnel and the precipitate was canceled then the filterate was concentrated to a quarter of its volume. After that 5 ml of concentrated sulphuric acid was added into the filterate and the contents were shaken well then the pH was adjusted to 9 by adding ammonia .The mixture was put in separation funnel and 20 ml of chloroform was added into mixture and contents were shaken well. Then extraction process was used and two layers were formed then organic layer volume was collected and aqueous layer was kept . This process was repeated by adding chloroform (20*3) to aqueous layer and organic layer was collected other time then the total volume was dried and the solid crude was gotten [15] with yield equal to 2.35 gm.

Gas chromatography -Mass Spectroscopy Technique
The alkaloides isolated from Alllium porrum seeds were separated then characterized by using gas chromatographymass spectrum procedure in GC-MS laboratory in college of agriculture in Basrah university by instrument type shimadzu-GC-MS-QP-2101 Ultra.

Stain of pathogenic bacteria
The pathogenic bacteria isolate represented by Staphylococcus aureus was gotten and identified by a microbiologist in microbiology laboratory -marine science center in University of Basrah Iraq.

Preparation of culture medium
The pathogenic bacteria used in this study was treated with Muller Hinton Agar (MHA) as a culture medium for growth of this pathogen.

Estimation of Medicinal efficacy of alkaloidic compounds
To evaluate the medicinal and biochemical effect of alkaloids isolated from Allium porrum seeds ,the method of Agar well diffusion was carried out . Two ml of Muller Hinton Agar was added into each petri dish then the medium was treated with 0.1 ml of pathogenic suspension (Staphylococcus aureus) has optical density equal to 0.1 with wave length is 450 nm by using spectrophotometer by using a sterilized glass spreader. Then the dishes were let for 25 minutes after that many wells were done and alkaloidic mixture was added with concentrations equal to 50,100,125,150,200,250,300,375,400 and 500 mg/ml and dishes were incubated at 37 C for 24 hours in the incubator. Finally the diameters values of inhibition zones were measured [16]

Results and Discussion
The health importance of Allium porrum plant indicates and proves the medicinal benefit resulting from existing of various active metabolites as natural drugs were used as antibacterial, antifungal, anticancer, antitumor and antiphrastic agents [17] [18]. Aqueous and alkaloidic extracts were prepared with weights equal to 6. 25  It is noticed that the extraction percentage of cold aqueous extract is higher than the the alkloidic extract so this biochemical state illustrates presence of more active metabolitic compounds in the aqueous extract than alkaloids . Table 2. shows the primary qualitative analysis of cold aqueous extract of Allium porrum seeds by using various chemical reagents depending on the chemical nature of secondary metabolites .  [20] . Alkaloids as secondary active materials were isolated by using different polar solvents and these potent compounds were gotten from the organic layer and this process insures       [26] .
Also the synergistic interaction of natural alkaloidic compound is chemically very powerful against the action of multi-enzymes abundant in the living cell of pathogens including Staphylococcus aureus by hydrogen bonding with thiol (-SH) group present in these enzymes leading to break the sulphuric bridges then denaturation of chemical nature belonging to these enzymes [27][28] .

Conclusions
The synergistic interaction of the natural alkaloidic mixture containing four active chemical compounds represented by 2-piperidinone, 2-pyrrolidinone-4-methyl, adenosine-2-deoxy-N-methyl -N-(triflouro acetyl) and maymyrsine isolated from Iraqi Allium porrum seeds showed the high chemical ability and medicial efficacy for inhibition of biological and physiological activities of Staphylococcus aureus pathogen causing multidiseases . Also it was proved that the increase of concentration of alkaloids gave more inhibition diameters values leading to kill most of the colonies of this bacteria. So the natural mixture of alkaloidic compounds of leek can be carried out as plant therapy against this pathogenic micro-organism and comparison of the medicinal efficacy of alkaloids characterized from this plant with antibiotics which target Staphylococcus aureus bacteria.