Phytochemicals screening and antimicrobial activities of selected medicinal plants of Khyberpakhtunkhwa Pakistan

The study was carried out to assess the phytochemical and antimicrobial bioassay of five medicinal plants, Lepidium sativum, Nerium oleander, Ranunculus repens, Tecoma stans and Urtica dioca. These plants are traditionally used as medicine in the Northwest Pakistan, therefore it is necessary to identify and estimate their alkaloid, flavonoid, saponin, phenol and tannin contents. Phytochemical inveigation of plant samples determines that alkaloid (63.6%) and flavonoid (0.91%) were highest in N. oleander, saponin (11%) and phenol (0.031) in T. stans, tannin (0.61%) in L. sativum, All five species showed no significant antimicrobial activities.


INTRODUCTION
The world is fertile with natural and medicinal plants.Medicinal plants are now more focused than ever because they have the capability of producing many benefits to society indeed to mankind, especially in the line of medicine and pharmacological.The medicinal power of these plants lies in phytochemical constituents that cause definite pharmacological actions on the human body (Akinmoladun et al., 2007).Phytochemical, natural compound occur in plants such as medicinal plants, vegetables and fruits that work with nutrients and fibers to act against diseases or more specifically to protect against diseases.
Infectious diseases are the leading causes of death through out the world that accounts for nearly one half of all death in the tropical countries, which are also becoming a serious problem in developed countries.It is calculated that infectious diseases are the main causes of death in 8% of the 9 deaths occurring in United States (Demissew and Dagne, 2001).In addition, antibiotics are sometime associated with adverse effects including hypersensitivity, immuno suppressant and allergic reactions.Given the alarming incidence of antibiotic resistance in bacteria of medical importance, there is a constant need for new and effective therapeutic agents.Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases from medicinal plants.Lepidium sativum, Nerium oleander, Ranunculus repens, Tecoma stans and Urtica dioca are medicinally very important plants and use extensively in pharmaceutical formulations and are also use by local practitioners for a variety of human diseases.Hence the aim of this study was to determine the phytochemical constituents and to investigate the antimicrobial properties so as to ascertain their uses in traditional medicines.

Preparation of sample
The aqueous extract of each sample was prepared by soaking 10 g of powdered samples in 200 ml of distilled water for 12 h.The extract was filtered through Watt man filter paper.The phytochemicals in each sample was determined qualitatively and quantitatively (Krishnaiah et al., 2009;Mattila et al., 2007).

Alkaloids
The extracts were evaporated to dryness and the residues were heated with 2% Hydrochloric acid on a boiling water bath.The extract were cooled, filtered and treated with the Mayer's reagent.The sample was then observed for the presence of yellow precipitation or turbidity (Tyler andHerbalgram, 1994, Harborne et al., 1973).Flavonoids 1.5 ml of 50% methanol was added to 4 ml of extracts.Warmed the solution and metal magnesium was added.Then added 5 to 6 drops of concentrated hydrochloric acid to the solution and observed for red coloration (Tyler and Herbalgram, 1994;Harborne et al., 1973).

Tannins
To 0.5 ml of extract solution, 1 ml of distilled water and 1 to 2 drops of Ferric chloride solution was added, observed for blue or green black coloration (Tyler and Herbalgram 1994;Harborne et al., 1973).Phenol 2 ml ethanol was added to the test solution and few drops of ferric chloride solution and observed for coloration (Tyler and Herbalgram, 1994;Harborne et al., 1973).Saponins 2 ml of distilled water was added to 2 ml of the test solution and shaked well and observed for frothing (Tyler and Herbalgram, 1994;Harborne et al., 1973) Quantitative analysis Alkaloids 5 g of the plant sample was prepared in a beaker and 200 ml of10% CH3CO2H in C2H5OH is added to the plant sample.The mixture HUSSAIN et al. 747 is covered and allowed to stand for 4 h.The mixture was then filtered and the extract is allowed to become concentrated in a water bath until it reaches ¼ of the original volume.Concentrated ammonium hydroxide was added until the precipitation is complete.
The whole solution is allowed to settle and the precipitate is collected and washed with dilute ammonium hydroxide and then filtered.The residue is alkaloid, which is then dried and weighed.

Flavonoids
Extracted 10 g of the plant sample with 100 ml of 80% aqueous methanol at room temperature.The whole solution was filtered and the filtrate was then transferred into a water bath.The solution was evaporated to dryness and weighed to a constant weight (Williamson and Manach, 2005;Mattila and Hellström, 2007).
Saponins 20 g of each ground plant samples were put into a conical flask and 100 ml of 20% ethanol was added to the plant sample.The said sample is heated over a water bath for 4 h at about 55°C with continuous stirring.The extracted mixture is then filtered and the residue is then re-extracted again with 200 ml of 20% ethanol.The collective residues are reduced to 40 ml over a hot water bath.The concentrated is then transferred to a separating funnel and 20 ml of diethyl ether is added to the plant extract and the shaken vigorously.The aqueous layer was recovered while the organic layer was discarded and the process of purification was repeated.Sixty milliliter of n-Butanol was added and combined n-Butanol extract were washed twice with 10 ml of 5% sodium chloride.The remaining solution was then heated on water bath and after evaporation; the samples were dried in oven to a constant weight.
mg of plant sample was weighed and transferred to 50 ml flask.Then added 50 ml of distilled water and stirred for 1 h.Sample was filtered into a 50 ml volumetric flask and the volume was made up to the mark.5 ml of the filtered sample was pipette into test tube and then mixed with 2 ml of 0.1 M ferric chloride.The absorbance was measured using spectrophotometer at 395 nm wavelength within 10 min (Tyler and Herbalgram, 1994;Harborne et al., 1973).

Phenols
Plants sample was boiled for 15 min with 50 ml of (CH3CH2)2O.5 ml the sample was pipette into 50 ml flask, and 10 ml of distilled water was added.Then 2 ml of NH4OH solution and 5 ml of concentrated CH3 (CH2)3CH2OH was added to the mixture.The sample was made up to the mark and left to react for 30 min for color development and measured for 505 nm wave length using a spectrophotometer (Tyler and Herbalgram 1994;Harborne et al., 1973).

Antimicrobial activity
Preparation of crude extract 100 g of each of the coarsely powdered plant material was taken and extracted with ethanol, water and n-hexane.The extracts was filtered and sodium chloride solution was then added to the filtered extract to form precipitates.The precipitates were then separated, dried and transferred to air tight amber glass container.The crude extract was dissolved in chloroform and water to make the final concentration, which was kept in refrigerator till use (Harborne et al., 1973).

Test for antibacterial activity
The antimicrobial activity of the prepared extracts was determined by using well agar diffusion method.The standardized bacterial stock suspension (10 8 to 10 9 ) colony forming units per milliter was mixed with 60 ml of sterile nutrient agar thoroughly.20 ml inoculated nutrient agar was poured into sterile Petri dishes.The agar was left to set and four well 10 mm in diameter was made in each of these plates using sterile cork borer No 8 and then agar discs were removed.The entire well was filled with 0.1 ml of each extracts using microtiter-pipette and allowed to diffuse at room temperature for 2 h.The plates were incubated at 37°C for 24 h.Two replicates were also performed for each extract against each of the test organism.Simultaneously addition of the respective solvent instead of extract was carried out as controls.After incubation, the zones of inhibition were measured and mean value was calculated (Hanna et al., 2008;Roberts and Wink, 1998).

Preparation of standard fungal suspension
The fungal cultures, Aspergillus niger (ATCC 9763) and Candida albicans (ATCC7596) were maintained on saboraud dextrose agar, incubated at 25°C for four days.The fungal growth was harvested and washed with sterile normal saline and the suspension was stored in refrigerator till used (Hanna et al., 2008;Roberts and Wink, 1998).

Test for anti fungal activity
The method applied for determination of antifungal activity is the same as for antibacterial activity.The media used is sterile yeast and mould extract agar.C. albican incubated for two days while A. niger for three days at 25°C.

RESULTS AND DISCUSSION
Phytochemicals are plant-derived chemical compounds which are non-essential nutrients, some of which show potential health-promoting properties.

Qualitative analysis
As can be seen from Table 1, alkaloids, flavonoids, saponins, tannins and phenols were present in studied plant samples.

Alkaloids
As can be seen from  in U. dioca.The use of alkaloid contains plants as dyes, spices, drugs or poisons can be traced back almost to the beginning of civilization (Roberts and Wink, 1998).They are well known for their CNS activities (Lewis and Elvin, 2003).

Flavonoids
Flavonoids are plant nutrients that when consumed in the form of fruits and vegetables are non-toxic as well as potentially beneficial to the human body; up till now, more than 2000 different flavonoids have been isolated from vegetables (Taiz and Ziegler, 2006).High percentage of flavonoids (0.91%) was determined in N. oleander followed by 0.60% in U. dioca, 0.53% in T. stans, 0.42% in L. sativum and 0.39 % in R. repens.

Saponins
Pharmacological activities have been reported about saponins such as antibiotic, antifungal, antiviral, hepatoprotective anti-inflammatory and anti-ulcer (Oakenfull, 1986;Zhang, 2001).Table 2 shows that the percentage of saponins (11%) was found very in T. stans, followed by 9% in N. oleander and 5% in R. repens.U. dioca contains 3.1% of saponins while in L. sativum the percentage was obtained in low concentration 2.8%.

Tanins
The concentration 0.61% was detected in L. sativum followed by 0.21% in U. dioca, 0.19% in T. stans while in the rest of samples, concentration of tannins was recoded 0.01%.Tannins are basically use for the treatment of inflammation, leucorrhoea, gonorrhea, burn, piles, diarrhea and as antidote in the treatment of alcaloidal poisoning (Buzzini et al., 2008).They are also used for tannin of animal hides to convert them to leather.

Phenols
Phenols are very wide spread in nature.They range from simple structures having a simple aromatic ring to highly complex polymeric structures and often exist in glycosidic forms (Williamson and Manach, 2005).Capsacin is found in the dried ripe fruit of different species of Capsicum.It had been used internally for dyspepsia and flatulence.Externally, it is frequently used as counterirritant (Mattila and Hellström, 2007).Table 2 shows very low concentration of phenols in the whole plant samples which range from 0.004 to 0.19%.

Antimicrobial assay
Sustainable amount of new antibiotic available in the market are obtained from natural or semi synthetic resources are obtained from about 20% of the plants present in world which were submitted to pharmaceutical or biological test.As can be seen from the analytical results obtained from the zone of inhibition of water extracts (Table 3) of five selected medicinal herbs

Water extract
Table 3 shows high activity 15 mm recorded from the crude extract of U. dioca against B. subtillis while less activity.10 mm was obtained from the crude extract of T. stans against B. subtillrespectivelyis.The high activity of L. sativum extract against P. vulgaris is 13 mm while 11 mm was recorded against the same bacteria in U. dioca of water extract.R. repens has showed 10 mm activity against P. valgaris.The activities of the other water extracts were relatively small; 9 and 8 mm was found in N. oleander and T. stans extracts.T. stans and N. oleander showed equal activity of 12 mm against S. aureus while less activity of 7 mm was recorded in R.
repens.An equal activity of 6 mm was observed in N. oleander and U. dioca against E. coli followed by 5, 4 and 2 mm in R. repens, T. stans and L. sativum, respectively against the pathogen E. coli.High activity of 4 mm was shown by L. sativum against P. aeruginosa while 3 mm activity recorded in N. oleander against same bacteria and same relatively low activiat 6 mm against S. typhi while 4 mm activity was observed from R. repens.Low activity of 2 and 1 mm was shown by N. oleander and L.

Chloroform extract
Table 4 shows high activity of 17 mm determined in the crude extract of U. dioca from chloroform extract against B. subtillis while the activity shown by the rest of the plant extracts were in between 9 to15 mm.Activity of 16 mm was recorded in the extract of L. sativum against P. vulgaris and 11, 10, 7 and 7 mm activities were found in U. dioca, R. repens, N. oleander and T. stans, respectively.R. repens extract showed 15 mm activity against S. aureus while less activity was found 9 mm in extract of L. sativum.Activity of 7 mm was recorded in T. stans extract against E. coli.5 mm activity was recorded in both crude extracts of N. oleander and U. dioca.While 3 and 2 mm activities were also observed.Extracts of N. oleander and L. sativum were more active, 4 mm against P. aeruginosa while T. stans and U. dioca also showed same activity, 3 mm and no activity was seen of R. repens against same organism.Activity of 7 mm was shown by L. sativum against S. typhi, 5 mm by R. repens, 2 mm by N. oleonder, 1 mm by U. dioca and no activity by T. stans against S. typhi.Same antifungal activity of 2 mm was determined against A. niger by most of the plants except R. repens which was 1 mm.T. stans was noted more active at 4 mm against C. albicans followed by 2, 1 and 1 mm in N. oleander, L. sativum and T. stans respectively while no activity was shown by R. repens.

Table 1 .
Qualitative analysis of phytochemicals.

Table 2 .
Quantitative analysis of phytochemicals.

Table 2
, high concentration 63.6% of alkaloids was found in N. oleander and less concentration of 0.40% was noted in L. sativum .The concentration of alkaloids in the rest of samples are as follow: 51.5% in T. stans, 0.80% in R. repens and 12.8%

Table 3 .
Zones of inhibitions of water extracts of selected medicinal plants.

Table 4 .
Zones of inhibitions of chloroform extracts of selected medicinal plants.while U. dioca showed no activity.L. sativum and U. dioca showed high activity of 3 mm against A. niger while 2 mm activity was found from R. repens and T. stans extracts while N. oleander showed no activity against A. niger.High activity of 2 mm was noted in both L. sativum and T. stans against C. albicans whilet same 1 mm activity was recorded in the rest of the plant extracts. sativum