Brine-Shrimp Lethality Bioassay of different extracts of the medicinal plant matricaria (chamomilla) owers

The present study aimed to throw some light on phytochemical constituents of Matricaria (cammomile) owers. The grinded owers were extracted successively by petroleum ether (40-60°C), chloroform, 95% ethanol, and ethyl acetate; using soxhlet apparatus respectively. The extracts were concentrated under vacuum (rotatory evaporator).Phytochemical screening of matricaria owers, were performed using standard methods, showed the presence of alkaloids, avonoids, terpenoids, saponins, and anthraquinone. The 95% ethanolic crude extract was subjected to column chromatography and nine fractions were collected. Fractions 1-6 have the same Rf value, (using thin layer pre-coated silica gel (0.25cm). were combined and subjected to spectroscopic analysis; infrared spectra showed absorption at 1860, 1637 and 805 cm’ indicating the presence of c=o, c=c, and c-o groups respectively. Ultra violet absorption at 250-283nm assign for the presence of avonoids, (lit. 250-280). Finally GC/MS spectra performed the presence avonoids.Crude extracts (ethanol, methanol, petroleum ether, and chloroform) were investigated for antimicrobial activities against four strains of bacteria (Staphylococcus aureus, Escherichia coli, Bacillus sub tills, Pseudomonas aeruginosa, and two fungi: Asperigillus niger, and Candida albican) with disc diffusion method, the diameter of growth inhibition zone ranged 15-22mm.The distilled essential oil (hydro distillation) of matricaria owers was subjected to GC/MS, a Number of aliphatic compounds were detected e.g. 2, 3 heptadione and 3-hexa none, 2, 5 dimethyl.


Introduction
Natural products from plants are important sources of drugs as several important medicines have been isolated and produced to cure ailments [1][2][3][4][5].
The pharmacological evaluation of extracts of organisms and pure isolates is an area of an essential aspect of the drug discovery process and developments in the techniques have substantially transformed this fact of natural product chemistry [1&3]. "Secondary testing" procedures need more detailed testing in order to select compounds for clinical trials [6]. The main needs of primary bioassay screen should meet the following: 1. The bioassay results should predict some type of therapeutic potential 2. Potentially useful pharmacological activity should not go undetected even though the activity may be either unexpected or unique.
3. The expected nature of the activity should be indicated for further research 4. The primary bioassay screening test should be adapted for impurities present in a crude extract.

5.
The bioassay procedure should be unbiased and it should allow for the coding of all samples, including both "known reference materials (standards) and unknown test samples and the results obtained should be repeatable.
6. The use of crude materials and pure isolates should be carefully studied.

7.
The single bioassay screen should as minimum as possible (1-2g) of the crude dry natural material (plant or animal extract).
8. Avoid expensive equipment or a sophisticated laboratory environment The procedure should be compatible with the use of dimethyl sulfoxide (DM50).

9.
The procedure should be simple enough to be taught easily to laboratory technicians [6] so that highly trained and qualified researchers are not required for the routine operation of the bioassay program [6&7] The choice of the screening approach to be adopted generally depends on the target disease as well as on the available information about the target organism (plant, marine animal, etc.) to be studied. For example if a plant has a ethanopharmacological history of use against a particular disease, then one would logically use a specific bioassay technique (single goal screening) which can predict the reputed therapeutic activity in order lead which is responsible for that biological activity[7&8].
Chamomile (Matricaria chamomile L.) is a well-known medicinal plant species from the Asteraceae family often referred to as the "star among medicinal species." German chamomile is used in herbal medicine for a sore stomach, irritable bowel syndrome, and as a gentle sleep aid. It is also used as a mild laxative and is anti-inflammatory and bactericidal. It is Acaricides used against certain mites, such as Psoroptes cuniculi. [9]. MatricariaL., 1753 Food and Drug Administration (FDA).Approved agent and continue to be one of the major source of inspiration for drug discovery [10&11].
Natural products may be extracted from plants, a crude (untreated) extract from any one of these source typically contains novel, structurally diverse chemical compounds [1, 12 &13].
Essential oil of chamomile was shown to be a potential antiviral agent against herpes simplex virus type 2 (HSV-2) in vitro P-Potential risks include interference with warfarin and infant botulism in very young children [14].
The present work focus on studying the phytochemical constituents of Matricaria (cammomile) flowers with different extracts with reference to brine shrimps.

Materials And Methods
The method described under the heading of brine shrimp lethality assay. The assay reliability correlates with KB cell toxicity assay. The presence of cytoctoxic natural products may be detected during the fractionation and isolation of natural product [15&16].

Results
1. Artificial sea water is prepared by dissolving sea salt in distilled water (40g/lii) supplemented with 6 mg/lit, dried yeast.
3. Brine shrimp larvae (nauplii) are collected with a Pasteur pipette are anring the organisms to one side of the vessel with a light so ire\auplii are separated from the eggs by pipetting them 2-3 times in small beakers containing sea water.
4. The test sample 2O mg of crude extracts or 4 mg for pure compound) is made up to lmg.ml in artificial sea water (water insoluble compounds or extracts can be dissolved in 5 m DM50 prior to adding sea water).

5.
Serial dilutions are made in wells of 96-well in triplicates in 100 P1 sea water ( Figure 1) and Table 1 6. A suspension of nauplii containing 10-15 brine shrimp larvae (100 ml) arc added to each well with the help of a Pasteur pipette and the covered microwell plate incubated at 22-29°C for 24 hours.
7. The plates are then examined under a binocular microscope (I 2.5x) and the number of dead (non-mobile) nauplii in each well counted.
8. 100 p1 methanol is then added to microwell and after I 5 iii in tcs the total nuin her of annaips in each well is counted.
9. LC50 values are then calculated by using prop analysis. 8. Test sample (crude extract, pure natural product or synthetic compound the chemicals applied were given in Table 2). Small amount of powdered drug is moistened with calcium hydroxide and distilled water to form a smooth paste and then extracted with chloroform. The chloroform layer is separated and evaporated to dryness on a water bath the residue is dissolved in 2 ml of methanol .0.1 ml is spotted by using capillary tube. The coating materials were usually applied as aqueous slurries. Slurry was made by mixing 30g of silica gel G with 60 mL of distilled water in a motor until it was of uniform consistency and free of air bubbles. The slurry was spread using a dragendroff's reagent spreader at 0.25mm thick layer on five glass plates (20x20 cm) The coated plates were dried at room temperature, then placed vertically in an oven and activated by heating to 10 0 C for 30 minutes.
Column chromatography (CC) Column chromatography was performed on a glass column packed with silica gel. The extracts were chromatographed after being absorbed onto a small amount of packing material or applied to the top of column by pipette.
Phytochemical screening of Matricria chamomile indicated the presence of secondary metabolites give positive result for Alkaloids , Flavonoids , Antraquinones ,Steroids give Tannins , Saponins and Glycosides as expressed in Table 1.

Anti-bacterial activity
The ethyl acetate extract is best solvent gave positive result for all types of bacteria , especially staphylococcus aureus as shown in Table 3.

Thin layer Chromatography
The chloroform and ethyl acetate extracts gave the best compound separation for Matricria chamomile .Concentrated ethanol extract isolated three avonoids compounds (Figure2).

Spectral data
The maximum absorption for three compounds at peak with RF ( Figure 3): The present study focused on the larvicidal as well as the Matricaria chamomilla activity of crude plant extract, comparatively more effective than other mosquito larvae of Matricaria chamomilla. In a study of Arivoli and coworkers [17 &18] had reported that larvicidal property of this .ant in ethyl acetate extract against Brine Shrimp larvae with an LC 50 value of 1.63 g/L. The comparison of safety two method the mosquito larvae was found to be signi cantly effective method to detect the Matricria Toxicity as a cheap and available method.

Conclusion
The phytochemical screening of Matricria chamomile owers re ect the presence of avonoids glycosides, tannins, anthraquinones, triterpense and sterols, saponins and alkaloids. Toxicity was carried out with Brine Shrimp, in comparison of safety two method the mosquito larvae was found to be signi cantly effective method to detect the Matricria Toxicity as a cheap and available method Declarations