Phytochemical screening and antimicrobial activity of selected medicinal plant species

Phytochemicals are essential compounds, utilized worldwide for curing of various human disorders. The present study comprised of 12 different medicinal plant species i.e. Withania coagulans, W. somnifera, Cannabis sativa, Medicago sativa, Achyranthes aspera, Convolvulus arvensis, Solanum nigrum, Mentha longifolia, Mentha spicata, Tagetes erecta, Fagonia cretica and Acacia nilotica. These species were used by the local inhabitants for treating various aliments. Methanolic extract of leaves of these plant species were investigated for cardiac glycosides, alkaloids, tannins, saponins, flavonoids, terpenoids, anthraquinones and reducing sugars. Among the reported medicinal plant species, Leaves of Acacia nilotica and Withania somnifera were tested for antibacterial activity which showed significant activity against Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas fluorescens.

In recent years drug resistance to human pathogenic bacteria has been commonly reported from all over the world [3].A large number of drugs are obtained from plant and used for many serious disorders [4].In East Asia, many plants are considered to have significants medicinal features i-e antiinflammatory, anti-bacterial and analgesic funcation because they contain a large variety of phytochemical i-emoniterpenoids, sesquiterpenoids, and curcuminoids [5, 6].Withania somnifera (family Solanaceae) is a medicinal plant utilized for large number of diseases.Every part of these plant have a big sources of many chemicals and widely used for cureing ulcers, fever, cough, dyspnoea, dropsy, rheumatism, toxicosis and leucoderma [7].Acacia nilotica.Is very common small tree it also known as kikar belongings to the family Mimosaceae [8].Acacia nilotica is consider to be antidysentric and antispasmodic .The pod and leaves are also utilized for medicinal purpose i-e diarrhea.Acacia nilotica part extract has been shown to exhibit antibacterial, antiplatelet and anti-oxidant.
[9].The stem bark of Acacia nilotica is well-known for its diuretic properties [9] These medicinal plants are important because of their uses for curing disorder and for healing as well [10].These plants are important because of their phytochemical and large number of significant features.There are many other reports which show importance of these plants for their antimicrobial activity and phytochemicals like [10][11][12].In Pakistan 6000 plant species were present In which about 180 plant were used for medicinal.The phytochemicals are two type including primary phytochemical which have not such a strong action in medicine while the secondary phytochemical are worldwide used for many ailments, the most important secondary phytochemical are Alkaloids, Terpenoids [12].Secondary phytochemicals are compounds which show no function to be incorporated in primary metabolism, they act as accessory role.Rather than medicinal uses these phytochemicals are used by the plant for their defense as well [12].Terpenoids play a vital role in most pharmacological work and activities, medicinally it play an important role in anticancer, anti-malarial and anti-fever as well [3].Alkaloids are also important secondary chemical which play a vital role as anesthetic agent.Alkaloids also create variation in physiology [13].Phenol and its compound are distributed in many plants, phenolic compound are used for the treatment of many disorder and also due to its toxic action prevent the growth of many pathogens [14].Flavonoids, sterol, and saponins are essential secondary chemical present in above 85 families, due to their multipurpose and specific chemistry they are used for the treatment of many disorder [15].All these phytochemicals give a strong immunity in the form of resistance against many insects and herbivores as well [16].In this universe about 4,44000 flowering plant species are present, in which 40000 plant species are utilized due its phytochemical importance.

Collection of plants
For the collection of plant species field survey were occurred throughout the research site during 2014.The listed plant species (Table 1) were collected and then dry.These plant species were identified at department of Botany Islamia College University, Peshawar.

Materials extract from plants
The dried material were obtained from these plant by keeping them at room temperature 28 degree celcius for 18 days, they were grinded into fine powder.Extract of ethanol were made through soaking 70g of powder from plant specimen in 1.3 L ethanol.For making a viable extract of ethanol 42 hours were required at room temprature.

Extraction of solvent
The dried leave of two plants i.e Withania somnifera and Acacia nilotica were washed and the slected plant material were dried.The parts of these plants were kept in oven at 40 °C then changed into powdered with mortar and pestle.80 grams of each parts of plant and 200 grams ml of methanol were kept in mortar and with the help of pestle it grind up .thisextract were placed at room temperature for 30 hours.This extract where filter through filter paper and dried at temperature below 48°C for methanol removal to obtain the dense extract and then they were kept in sterile bottles under refrigerated conditions until use.

Anti-bacterial testing
Antibacterial activity was measured using agar dilution technique.Briefly, the methanol extracts were dissolved in dimethyl sulfoxide (DMSO, Merck) and serially diluted in molten Mueller Hinton Agar (MHA, Sigma) in petridishes (100 mm×15 mm) to obtain final concentrations: 100, 50, 25 and 12.5 µg/ml.The solvent did not exceed 1% concentration and did not affect the growth of the organisms.All bacterial strains were grown in Mueller Hinton Broth (MHB, Sigma) for 4 h at 37°C.Bacterial suspensions with 0.5 McFarland standard turbidity, which is equivalent to 108 cfu/ml were prepared by dilution with Mueller Hinton broth.The diluted inoculum was added to a Steer's replicator calibrated and incubated for 24 h at 37 °C.After incubation, all dishes were observed for microbial inhibition by the disc diffusion method (Table 2).Rotary Evaporator By using rotary evaporator the extract become concentrated.The solutions were also passed through filter paper to make it impurities free and the analyses of phytochemicals are occurred by some procedures which are standrad.

Cardic glycosides test
Cardic glycosides test needs chemical like Ferric chloride, glacial acetic acid and distal water as well.Extract of 0.7 gm diluted through 10 ml of water, 3 ml of glacial acetic acid and also few drop of feric chloride were also included.Specific colors were responsible for sugar indicator and light brownish spots were indicative feature of deoxysugar, also some green spots were present as well.

Flavonoids test
For the determination of flavonoids dilute amonia was introduced.3ml of dilute amonia were added to extract, sulphiric acid were also used.By the appearance of yellow colour showed that presence of flavonoids [17,18].

Alkaloids test
For the indication of phytochemical like alkaloids, 0.6 g of extract were mixed to 7ml acidified alchole, after mixing they were boiled and then their filteration occurred.4ml after filteration was mixed with 2ml of dilute amonia, 3ml chlorofome were introduced and then mixed, after mixing gently the chlorofome appearance occurred in the form of layer with 6ml of acitic acid.At last the formation of brownish red precipate appear which clearly indicate alkaloids [19,20].Terpenoid test Take 0.3g of the sample extract and add to 2ml of chloroform.A small amount of concentrated H2SO4 (2ml) were introduced for obtaining the phytochemicals.The appearance of reddish brown colour showed the presence of terpenoids.

Tannins test
Boiled extract about 0.3g in 7ml of water were mixed in a test tube and then filter.Ferric chloride was also add about few drops 0.2% after addition blue black or brownish green colour occurred [19,20].

Anthraquinones test
Boiled extract about 0.3 g were taken and mixed with 8ml of sulphuric acid after mixing then filtered.Mixed 4ml of chloroform with filtered and then shaked a layer of chloroform were formed which was pipette into test tube at last 2ml of ammonia were add and wait for colouration.Reducing sugar test Take 0.3g of sample and add 7ml of water.This mixture was add to boiling fehling's solution after reaction colour was observed.

Saponins test
Test for saponins required oil of olive, 0.4g of extract was taken and then mixed with 7ml of water and make a solution, this solution was mixed with a few drop of olive oil.Observed the colour [21,22].

Test sample preparation for antimicrobial test
For the antimicrobial tests, ethanolic extracts were diluted in dimethylsulfoxide (DMSO): methanol (1/1: v/v) solvent to a concentration of 20 mg/ml.
Introduction There are some diseases in the world which cause death, almost of 40,000 people.Disease like diarrhea cause huge mortality among childrens [1].Bacteria like Escherichia coli, Salmonela spp.and Staphylococcus aureus are most common species which are pathogen on children [2].

26-28] also
[30,31]d the antimicrobial activity of of these flora which were utilized in making medicine same is true in the present finding.Withania somminfera is another important medicinal plant species containing many important alkaloids which are used in medicine[29].[30,31]alsoreported Acacia nilotica and Withania somnifera showed significant antibacterial activity against Bacillus subtilis, Escherchia coli, stphaylocuccus aureus and pseudomonas fluorescence.Same is true in the present finding.