In- Vitro Study of Antibacterial Properties and Phytochemical Contents of Coriander Seeds Against Five Bacteria.

In an effort to highlight the role of digestion and maceration methods to determine the strength of antibacterial effect and phytochemical elements of coriander seeds aqueous and methanolic extracts. Five concentrations (20, 40, 80, 160 and 320 mg/ml) of four extracts (aqueous by maceration, aqueous by digestion, methanolic by maceration and methanolic by digestion) were utilized against five clinically isolated bacteria (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aerogenosa and Acenitobacter baumani), and compared with ciprofloxacin and cefotaxime discs as control. Agar well diffusion technique was employed to clarify the antibacterial activity of the extracts' concentrations relaying on quantifying the diameter of inhibition zone (IZ) in millimeter. The bioactive chemical compounds of four extracts were specified by means of conventional systems. In the main, all extracts inhibited the growth of every bacterial species starting from (80) mg/ml concentration, forming IZs' with various measurements that their expansion was drawing upon the rising of extracts' concentrations. The IZs that proceeded from (160, 320) mg/ml concentrations were larger than that come out from cefotaxime. The maceration extracts declared their embracement of alkaloids, and their impact were vigorous compared with the extracts of digestion which marked by comprising the tannins and flavonoids in aqueous and methanolic extracts one by one. The most susceptible bacterium to ethanolic extracts was S. aureus. It could be benefiting from coriander seeds for treatment of bacterial infections through extraction the Kirkuk University Journal /Scientific Studies (KUJSS) Volume 14, Issue 2, June 2019 , pp. (1-18) ISSN: 1992-0849 (Print), 2616-6801 (Online) Web Site: www.uokirkuk.edu.iq/kujss E. mail: kujss@uokirkuk.edu.iq, kujss.journal@gmail.com 2 best active phytoconstituents by using both of suitable extraction technique and solvents together.


Introduction:
Multidrug resistant bacteria can distribute in human society, has significant risk on popular hygiene and must be controlled immediately and firmly [1]. It is essential urgently to develop very unique treatment plans and seek for new antibacterial medications that are further efficacious and has capacity to stand up seriously and persistently opposing to strategies of bacterial resistance [2]. In the present time, the real benefits such as plentiful experimental practices and existence of distinctive variety of chemical elements that possess biological properties are recorded from employing of natural products and indigenous medicine to frame modern remedies [3], especially detection of antibacterial remedies throughout persistent investigations which is considered the main basis of antibacterial work field [4]. Different plant extracts done by numerous solvents brought into view the hopeful antibacterial impact of such extracts contrary to human's pathogenic bacteria; this idea proved through number of researches [5]. The herbs contain variety phytochemicals exemplified by secondary metabolites which considered the origin of medications and can be attended as fresh antibacterial factor [6], owing to if compared with artificial antibiotics, they are harmless without side effects, efficient clinically, do not develop resistance and cheaper [7]. Coriander is a yearly herb which is belonging to carrot family (Umbelliferae) and Coriandrum genus. It consists of two species; the first is C. sativum, a broadly crop which is available primarily in the tropics, though the other species is C. tordylium which is recognized as uncultivated plant [8]. C. sativum is exactly named as "herb of happiness", on the strength of it looked at as one of the wonderful herbs because it consumed as spice along with herbal prescription together [9], including different employing it for different intentions for instance: in nutrition foodstuffs as flavoring material, covered commodities, brews, cosmetics, tobacco yields, perfumes, and as principal factor for curry powder [10].
There are surveys that elucidated biological action of all segments of Coriandrum sativum for example fruit, flower, seeds and leaves; these action are antidiabetic, antioxidant, diuretic, antimutagenic, hypnotic, sedative, anti-convulsing, anthelmintic and antimicrobial [11], attributable to existence of different chemical constituents such as alkaloids, glycosides, reducing sugars, tannins, phenolics, flavonoids, essential oil, terpenoids, sterols and fatty acids [12].
On other hand the antibacterial potent of C. sativum has been established by several reports; the colonies of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aerogenosa and Salmonella typhi was affected at numerous marks by stems and leaves of coriandrum extracted by four solvents (acetone, ethyl acetate, methanol and petroleum ether) as a general rule [13]. Fazeel verified the antibacterial activity of C. sativum essential oil against P. aerogenosa, S. typhi, E. coli, Klebsiella pneumoniae and S. aureus, the oil suppressed the culture of all prior bacteria preferable off than or nearby the antibiotics [14].
The existing research was directed to recognize whether there are variations between maceration and digestion extracts approaches employed to prepare aqueous and methanolic extracts of C. sativum seed as antibacteria against five selected bacteria in vitro depending on exploring the selected phytochemical ingredients of each extract.

Materials and methods:
2.1 Practiced plant:

Plant sample:
Coriander (C. sativum) seeds were bought from herbal merchant in Kirkuk city during august 2017, and then were delivered to laboratory technique department of Kirkuk Technical College.
Taxonomic approving of the plant sample was authenticated according to conservative means.
The choosing of the plant relying on its consuming as ethnic herbal medication.

Plant treating:
The seeds were eliminated from dirt by rinsing with distilled water. After aeration of seeds at room temperature, they were milled with electric grinder. The powdered seeds were stocked in nylon pouch at 4 °C.

Plant extraction:
Both of the aqueous and methanolic extracts of coriander seeds were prepared by maceration and digestion methods in conformity with [15]; as two bottles every one of them containing 500 ml of distilled water were readied for aqueous extracts and in other hand methanolic extracts were equipped with two bottles each one of them comprising 500 ml of 99 % methanol (Scharlab S. L., Spain), at that time 400 (g) of seed powder was balanced via the digital balance (Denver instrument, Germany) and thawed in four bottles (100 g in every single bottle). Two bottles (one of aqueous, the other of methanolic) were macerated for one weak at room temperature with regular jerking until the resolvable material has softened. The digestion technique was executed on the other two bottles (one of aqueous, the other of methanolic) the same as maceration procedure plus heating at 50° C in water bath for 24 hours, there filtration using muslin cloth was achieved on the extract and the superfluous solvent was eradicated from consequential filtrate by oven (Memmert, Germany) at 70 °C for 48 hours that lead to the foundation of a sticky substance which deposited in airtight sterile containers that left in refrigerator up to afterward screening.

Readying of extracts' concentration:
The following concentrations (20, 40, 80, 160 and 320 milligram (mg)/ml) of each aqueous and methanolic extracts (produced from maceration and digestion methods) were organized through following style: 200, 400, 800, 1600 and 3200 mg of aqueous and methanolic sticky substances were dissolved separately in 10 ml of distilled water. All concentrations were preserved in airtight cups at 4 °C awaiting of exploration them for antibacterial potential. The antibacterial impact of the concentrations was compared with distilled water as negative control, on the other hand positive control signified as ciprofloxacin and cefotaxime.

Primary phytochemical composition tests:
The overall extracts were exposed to primary phytochemical investigations for revealing many plant phytochemical components employing standard tests as reviewed underneath: 1-Showing alkaloids by Wagner's Test: Extracts were admixed separately in 1% dilute Hydrochloric acid and filtered, and then 2 ml of filtrates solution were supplemented with 6 drops of Wagner's reagent (Iodine in Potassium Iodide in 100 ml water). Observation of brown/reddish precipitate establishes positive test and the existence of alkaloids [16].

Description of bacterial isolates:
The following typical microbiology procedures mentioned by [19] were resorted the specifications of every bacterial species; colony morphology after culturing in enriched media then sub culturing in differential and selective media to detect the fine colonies, Gram's staining inspection, documentation the biochemical features of each species by way of initial biochemical tests.

Continuation of bacterial isolates:
Few pure colonies of every single bacterial species were sub cultured on nutrient agar which incubated overnight, later on repetition of sub culturing on nutrient slants were performed monthly as stock culture and conserved at 4 °C until analyzing the antibacterial activity of the extracts.

Making and normalization of bacterial inoculum suspension:
A loop filled with bacterial growth was picked up from stock cultures and immersed into nutrient broth with a view to prepare suspension for each bacterial isolates, the broths were

Antibacterial effect checking:
Under excessive aseptic conditions, the antibacterial specialties of coriander extracts toward examined bacteria were observed on the authority of agar well diffusion technique In vitro [20].
The bacterial suspension of each bacterium was spread out on surface and margin of nutrient agar plates by means of sterile cotton swab. After about 20 minutes the plates were hardened, thereupon the surface of every plate was punctured identically in six sites in order to create six mm diameter holes with cork borer. One hundred microliter (µl) from each extract concentrations was put in every hole, together 100 µl from distilled water was placed in one hole by utilizing micropipette [21]. Agar disc diffusion technique [22] was also carried out with a view to assess antibacterial susceptibility of ciprofloxacin and cefotaxime discs against isolated bacteria when the discs were resided at plates' surfaces with sterile forceps. Later on the antibacterial action was identified by way of noticeable inhibition zone (IZ) surrounded the holes

Statistical valuation:
Excel program of Microsoft office 2010 was utilized for appraising the results. Altogether the antibacterial effect of every one extract's concentration was represented by way of mean IZ diameter of three evaluations by millilmeter (mm) ± standard deviation (SD).

Results:
In this study as designated in Table 1 Table 3: Sensitivity of studied bacteria in the face of positive and negative controls.  Table 4. Table 4: Phytochemical searching of digestion and maceration methods extracts.

Discussion:
Observations achieved from current study revealed that coriandrum seeds aqueous and methanolic extracts from 80 to 320 mg/ml concentrations prepared from maceration and digestion methods have spectrum antibacterial action alongside all tested bacteria as whole Tables 1, 2  Results of present study is also not in agreement with investigations of Oudah and Ali, when  [21]. The activity of aqueous extract obtained by maceration method at 320 mg/ml toward P. aerogenosa was apparent by 9.33 mm IZ; this is seemed to be in line with the obtaining of 9 mm IZ by aqueous extract of coriander seed at 100% concentration upon the said bacteria [32].
Pursuant to results appeared in Tables 1, 2

Conclusion:
In assumption to the existing results, the coriander seeds aqueous and methanolic extracts seemingly suppress the growth of all tested bacterial species owing to the attendance of forceful phytochemicals in these extracts. The extracts got from maceration method are better inhibitory antibacterial than digestion method. Extraction practice considered to be essential step for successful segregation of chemicals from herbal substance as well as the category of extraction solvent. In recommendation, it is mandatory to check the best extraction method for detection and standardization the more active extracts accountable for antibacterial activity on numerous antibiotic resistant bacteria through the phytochemical compounds that originate in such extracts.