Phytochemical screening and antibacterial assay of the crude extract and fractions of Ferula oopoda

The principal objective of the current study was to analyse phytochemical constituents and to determine the antimicrobial activity of the crude methanol extract and fractions of chloroform, ethyl acetate and hexane from the whole plant of Ferula oopoda against three bacterial strains Escherichia coli, Salmonella typhi and Staphylococcus aureus. Phytochemical assay confirmed the presence of terpenoid, flavonoids, saponins, tannins, phenolic compounds, carbohydrates, steroids and glycosides. Agar disc diffusion method was used to determine the zone of inhibition of the tested sample for antimicrobial activity. The crude methanolic extract showed activity against E. coli ZOI, 30.00±1.060 mm, for ethyl acetate fractions 50.00±4.18 mm, for chloroform fraction 27.00±0.060 mm and for n-hexane fraction 24.00±0.353 mm. This observation shows that ethyl acetate fraction possesses great potential against E. coli. Inhibition zone for Salmonella typhi was 23.25±1.050 mm for ethyl acetate, 14.00±0.353 mm for crude methanol extract, 22.00±1.753 mm for chloroform fraction and 08.00±0.352 mm for n-hexane fraction. This observation shows that n-hexane fraction possesses low potential against Salmonella typhi. Anti-bacterial potential against Staphylococcus aureus strain was maximum in ethyl acetate fraction and showed ZOI, 34.00±1.767 mm, for chloroform fraction 21.24±2.636 mm, for crude methanol extract 19.00±1.060 mm and for n-hexane fraction 16.00±1.412 mm respectively.


Introduction
Infectious diseases are caused by pathogenic microorganisms; some organisms in extreme circumstances can be fatal to the host.According to WHO 80 % of the world's population uses plant extracts or their active ingredients as folk medicine in several traditional treatments [1].Plants are considered as a valuable source of therapeutic agents.The research based on the Indo-Pakistan subcontinent it was recorded that plant species were used for medication in Rigveda between 4500-1600 B.C.There is an extensive knowledge and research based applicability already exist in this region.Some native plants are still widely used in rural areas of Pakistan especially in Balochistan province due to their antimicrobial effect without any logical evidence [2].People use Ferula species for different medicinal purposes such as to get rid of gastric and intestinal worms, lowering blood pressure and controlling diabetes.The methanol extract of Ferula oopoda possess anti-plasmodial activity [3].The continuous use of plants in folk medicine makes it important to screen the medicinal plants for the discovery of new antimicrobial compounds.The growing concern about the resistant bacterial strains against the antibiotics is of focus point in international communities.Plant constituents having antimicrobial activity can hinder bacterial growth by different mechanisms as compare to that shown by currently used antibiotics.They may also have a significant clinical value in the treatment of resistant microbial strain [4].Among the plants of Balochistan, the Apiaceae family has a great representation, and several species are used because they contain antimicrobial compounds, antiinflammatory and antifungal agents [5].The Species Ferula oopoda belong to the family Apiaceae, subfamily Apioideae and genus Ferula.This plant is found in Balochistan region of Pakistan and also found in neighbouring countries of Pakistan such as Iran, Afghanistan and India etc.In Pakistan this plant is mostly cultivated in Northern areas and in Balochistan such as Ziarat, Harboi, Chautair, Chasnak and Sasnamana etc.This medicinal plant possesses amazing significance for the treatment of toothache and gastric disorders.Several biological activities have been described for the species of this genus, such as antibacterial, antioxidant, antifungal, anti-plasmodial and anti-inflammatory activities.These activities are often attributed to the presence of phytochemicals which are bioactive compounds also known as secondary metabolites [6].Phytochemicals include terpenoids, flavonoids, saponins, tannins, phenolic, carbohydrates, steroids, proteins, glycosides, their essential oils and some non-volatile compounds [6].These are produced in almost all parts of the plant like leaves, bark, stem, flower, root, seeds and fruits etc [7].Because of the scarcity of research studies about Ferula oopoda, the aim of this work was to comprehendon the first bioassayguided isolation of the extract and phytochemical investigation of the compounds, to carry out the antimicrobial activity of plant extracts and its fractions against certain pathogenic bacterial strains.

Plant collection and sample preparation
The plants were collected from different areas of Harboi (mountainous region of district Kalat, Balochistan, Pakistan).The species for this study was identified as Ferula oopoda.The whole plant was washed with the distilled water and dried naturally i.e. under shade.After completion of drying process, material was ground in a crusher and the powder was stored in sealed plastic bags for further analysis.

Extraction procedure
Solvent extraction method was used for extracting phytochemicals.Powdered plant sample was soaked in methanol, and then the mixture was subjected to rotary evaporator till the gummy extract was obtained after solvent evaporation.This extract was stored at room temperature for phytochemical and antibacterial analysis.

Fractionation of crude extract of methanol
Liquid-liquid extraction (LLE) commonly known as solvent extraction and partitioning, is a method where compounds are separated based on their relative solubility in two different immiscible liquids and separate into layers when shaken together.Solvents selected for the present study were ethyl acetate, chloroform and hexane.Selection of these solvent was based on polarity order.First, methanolic extract was dissolved in 200 ml of distilled water then poured in separating funnel followed by addition of 400 ml ethyl acetate solution.Layer between two different solvents appeared in separation funnel which was then sealed.
Funnel was shaken vigorously for 20-25 minutes and pressure was released at regular intervals.After 25 minutes two layers were separated, containing fraction in ethyl acetate and n-hexane solvent.The procedure was repeated twice and the obtained fractional extracts were placed in rotatory evaporator at 100 rmp speed to obtain gummy extract material of the fraction.These fractions were stored in refrigerator at -2 0 C. The methanolic extract was dissolved in 200 ml of distilled water then poured in separating funnel followed by addition of 400 ml chloroform solution and as mentioned above the same procedure was followed.The procedure was repeated twice.Obtained fractional extracts were placed in rotatory evaporator at 100 rmp speed to obtain gummy extract material of chloroform fraction.Exactly same procedure was followed to obtain n-hexane fraction.Obtained amount of fractional extracts for ethyl acetate was 0.8g, for chloroform was 0.65g and for n-hexane was 0.432g respectively.These obtained fractionations were used for phtochemical analysis, TLC, UV and FTIR spectroscopic analysis and antibacterial activity.

Phytochemical analysis Terpenoid test (Salkowski test)
Crude methanol extract (5 ml) was added 2 ml of chloroform and 3ml of concentrated sulfuric acid on the test tube's side wall.

Reddish brown colour shows the presence of Terpenoid [8]. Flavonoids test
Mixed methanol crude extract of plant with 3 ml ammonia solution followed by careful addition of sulfuric acid.Yellow colour appeared which indicated the presences of flavonoids in sample [9].

Saponins test (Foam test)
To the methanolic extract of the plant (0.5 ml) was added 20 ml of water.The mixture was shaken thoroughly for 15  Thin layer chromatography (TLC) preparation of TLC plates TLC plates coated with silica gel were used for the analysis.Each plate was of 30 cm length.A line was marked at 1.5 cm from bottom of TLC coated plate.

TLC separation
Mobile phase was prepared by mixing ethyl acetate, chloroform and acetone with ratio 2:2:1.Chromatographic tank was covered and left for some time before analysis so that all tank gets saturated by vapours of mobile phase [14].Capillary tube was used for spotting of plants extracts on TLC plates.The ultra violet (UV) lamp was used to visualize all spots appeared after separation on TLC plates were placed under the UV lamp and circles were drawn to visible zones and followed by Rf values calculations of different compounds.

UV-VIS spectroscopy
UV-visible spectrophotometric analysis was carried out at room temperature by using spectrophotometer (Perkin Elmer, USA Model: Lambda 950).The extract and fractions were examined under wavelength from 200-to-800nm and the wavelength selected for research was 300 nm.The crude extract of Ferula oopoda was diluted to 1:10 with distilled water.Distilled water was taken as blank and absorbance of the sample was measured and spectra were obtained.Same procedure was followed for chloroform ethyl acetate and n-hexane fractions [14].All gummy samples were first dissolved in methanol solvent and then placed in sample cuvettes.Reference cuvette was filled with dilute methanol solvent.Each fraction was tested one by one.In each case absorption was recorded.Fourier-transform infrared (FTIR) spectroscopy FTIR spectrometer was used for the identification of the characteristic functional groups in the extract and in the fractions.This spectroscopic technique provides structural information of a molecule from the obtained absorption spectrum.Very small amount of the sample was used for analysis.The spectrum was obtained using Bruker, Germany Vertex 70 infrared spectrometer.The wavelength range used for sample analysis was 4000 to 625 cm-1 [15].The spectrum was obtained and peak values were recorded Antibacterial activity For antimicrobial activity of an extract or pure compound a variety of laboratory methods can be employed.Broth/agar dilution or disk-diffusion methods are the most commonly used one [16].

Agar well diffusion method
The antibacterial activity was analysed for crude extracts and fractional extracts of selected medicinal plant.All samples were dissolved in dimethylsulphaoxide (DMSO) solution followed by filtration.The target bacterial species were refreshed in nutrient broth by inoculating them in nutrient broth and incubating at 37°C for 24h.The prepared bacterial culture were spread over the surface of sterile Mueller-Hinton agar plates using sterile cotton swabs.Well (6 mm) were bored in the media and 100 µl of each extract was poured aseptically.Plates were observed after 16-24 h incubation at 37 0 C. The extracts activities in term of zone of inhibition (ZOI) were analysed visually and recorded in millimetre [17].DMSO and Doxycycline (DO 30µg) were used as negative and positive control for this study.

Results and discussion
Ferula oopoda plant was screened for its phytochemical constituent and antimicrobial activity.Preliminary phytochemical analysis showed that the plant had considerable proportion of important phytochemicals that were detected by qualitative tests.From analysis it was cleared that Ferula oopoda is rich in flavonoids, steroid, glycosides, tannins and phenolic compounds (Table 1).From the literature survey it was found that flavonoids have a variety of biological properties such as antibacterial, antiinflammatory, antiviral, anti-allergic, cytotoxic antitumor properties.It is used in the treatment of neurodegenerative diseases and has vasodilatory action.It

TLC profile
The retention factors (Rf) of crude methanol extract, ethyl acetate and nhexane and chloroform fractions in solvents system are shown in (Table 2).The chromatogram revealed 3, 3, 2 and 2 spots for crude methanol, ethyl acetate, chloroform and n-hexane fractions respectively.TLC profiling of crude methanol extract, ethyl acetate and nhexaneand chloroform fractions was very impressive which directs towards the presence of a variety of phytochemical.Rf values of different phytochemicals differ in different solvent system.These variable Rf values provide important information about the polarity and selection of solvent system for chromatographic separation of pure compounds in column chromatography.Mixture of solvents of different polarity in variableratio can be used for separating pure compound of plant extract.Therefore, Rf values of compounds in variable solvent system can be used for selecting appropriate solvent system for plant extracts [21].Secondary metabolites of plants can be identified accurately by TLC method.The obtained different Rf values in this presented study showeddifferent profile in a single extract.The results also gave an idea about the polarity which helps during the selection of suitable solvent system for further separation of compound from the plant extracts.

FTIR analysis
The FTIR analysis was done for the identification of functional group of the constituent components on the basis of various peaks obtained in the IR region.
The FTIR spectrum of the Ferula oopoda plant extract is shown in (

Antibacterial assay
The antibacterial activity showed positive response in all fractions and in crude methanol extract (Table 5).

Conclusion
This study gave an insight and information for the determination of chemical composition of Ferula oopoda using different biochemical tests and results were confirmed by TLC, UV-VIS and FTIR techniques.In the present study flavonoids, steroid, glycosides, tannins and phenolic compounds were identified from methanolic plant extract and its different fractions.The presence of phytochemical constituents in Ferula oopoda gives credible evidence to its use by the human being.Novel drugs can be prepared by isolating these specific compounds.It could be concluded that Ferula oopoda contains various bioactive compounds which makes it an important pharmaceutical plant.However, further studies are needed to be undertaken to determinecomprehensively its toxicity profile, bioactivity, effect on the ecosystem and agricultural products.

Table 5 . Inhibition zones of Ferula oopoda methanol extract and different fractions against pathogenic bacteria.
The chloroform fraction, crude methanolic extract and n-hexane showed ZOI as 21 mm, 19 mm and 16 mm respectively.These observations reveal significant potential of plant to act as antibacterial agent.Thus, plant active component may be useful in synthesis of effective drugs to treat bacterial infection.