Phytochemical screening and antibacterial activity of neem extracts on uropathogens

The study investigates the antibacterial activity of leaves ethanolic extract of Azadirachta indica on human uro-pathogens that having the ability to cause urinary tract infections. The extract screened for S. aureus , E.coli and P. aeruginosa, for their antimicrobial efficiency. For this purpose, MIC, MBC and antibiotic sensitivity test were performed. Zones were appeared in agar well-containing leaf extract as compared to sensitivity test. The difference between MIC and MBC at tube 4 neem show the activity against E. coli and P. aeruginosa while for S. aureus results showed the inhibitory effect. The phytochemical analysis shows that neem has components which enhance the antimicrobial activity of neem. Phytochemical screening of neem not only having antimicrobial activity but also have antifungal, antiviral properties this activity of neem comes due to its bitter taste and the taste is due to the presence of components likes alkaloids, flavonoids, triterpenes. In current era the antibiotics therapies are more risky to cure infection because antibiotics has vast side effects that directly effects the certain body parts mainly kidney, stomach, upper and lower respiratory tract not only effects but also cause respective body organs disorder So, to subside the effects of antibiotics we are now able to cure microbial infections by the use of herbal medicine. In which NEEM has played a vital role to cure both infections skin as well as inner body infections.


Introduction
The emergence of multidrug-resistant organisms in the present era is becoming a big issue [1].This concept is changing now because of the use of the non-toxic medicinal plant.In order to manufacture effective drugs which can be able to treat such types of infections which can be initiated by the multi-drug resistant organisms [1].The versatile medicinal plant is neem (Azadirachta indica) which have several chemical and biological properties which are effective against infections.aswe know very well that the UTI urinary tract infection is a second most common infection of both males and females approx.8.1 million peoples carry this infection with or without symptoms each year (Lane and Thakkar).UTI is more common in females as compared to males.The most common cause is hospital-acquired UTI which is approximately 40% the age of women is a very important factor, 2-7 % cases reported in their fertility period, more high rate of cases reported at the age 40-50 that is 50%.and7-10% case reported in males at the age of 70-75.The infection is carried out when bacteria start to colonize in the bladder and weaken the urinary system [2].The number of bacteria may be 10, 0000/ml to cause infection.The most common pathogens are gram-negative including E.coli, Pseudomonas,Klebsiella,Proteus,Serratia,.90% of cases are caused by gramnegative organisms and 10% of cases reported by gram-positive bacteria [4, 5].The severity of infection start when the pathogens build resistivity against antibiotics by developing certain changes it may include the changing of the target site of drugs, destroying the antibiotics.This changes will help to makes the infections more severe and more fatal.[6] By using medicinal plant we can be able to treat the infection because it contains certain structure which helps to recover the infection.Neem is one of the oldest plants which can use as folk medicine.Neem is an evergreen tree cultivated worldwide; it was used as a therapeutic agent for certain infections.In the past era neem used to treat tooth decay and skin problems but nowadays neem also used to treat many infections including hair problems, ulcer, tumors, diabetes, abdominal pain, fever, cough, etc. Neem not only having properties to treat infections but also have anti-oxidant, anti-malarial, anti-carcinogenic, antimutagenic, and anti-inflammatory and antihyperglycemic properties [7].In the modern era, many pharmaceutical companies are engaged in discovering drugs by using medicinal plants like Neem.It is a versatile plant which shows antibacterial and antifungal properties as well as antiviral activity viruses like vaccinia, chikungunya, measles and coxsackie virus [8].The plant leaves not having antimicrobial properties but its bark, seeds, flower, fruit, and roots also having these properties [9].Neem is well known for their antibacterial property because it contains certain compounds which make the neem antimicrobial and due to these compounds neem's taste is bitter which real power against harmful microbes is.The powerful active components are alkaloids, flavonoids, glycosides, triterpenes, reducing sugars, tannins, saponins, and anthraquinones.This study aimed to investigate the antibacterial against uropathogens and phytochemical screening of neem ethanolic extracts [10].Neem not only helps to prevent the infections but also used in place of antibiotics.Antibiotics are very well known to cure the infections but also involved to destroy the immune system.When antibiotics kill the bacteria our immune system also damaged to avoid this type of damage we use medicinal plants to cure disease with damaging the immune system [11].

Collection of isolates
The clinical urine isolates were used in this research, were collected from the Department of Microbiology lab of Jinnah University for Women and also from the City Lab and Diagnostic Center.The isolates were then purified and stored.Total 3 bacterial strains were used.Two were gram-negative E.coli and Pseudomonas and remaining 1 was gram-positive S. aureus.

Preparation of plant extract
The neem extract was made in two different ways in ethanol and in distilled water to check the antibacterial activity.

Neem extract in methanol
Fresh leaves of Neem were collected locally and were air dried in shade.Before obtaining the extract first make the powder of leaves by grounding the leaves using a grinder and make a fine powder.Take 2 gm of neem leaves powder and add in 20 ml of ethanol mix well in 100 ml of the flask.Put the flask on a mechanical shaker for two days.After two days filter, this suspension by the help of watt's man filters paper 1 into another flask.Preserved the extract in brown bottle place the bottle in the refrigerator.

Neem extract in distilled water
Take 2 gm of neem leaves powder and add in 20 ml of distilled water mix well in 100 ml of the flask.Put the flask on a mechanical shaker for one week.After one week filter, this suspension by the help of watt's man filters paper 1 into another flask.Preserved the extract in brown bottle place the bottle in the refrigerator.

Agar well diffusion method
In 1 ml sterile saline add 4-5 colonies of respective cultures till the moderate turbidity was developed and then match with McFarland tube 0.5.The fresh inoculum was ready to use.Take sterile swab and dip the swab into the inoculum before removing the swab touch the swab to the wall of the tube so that the excess inoculum removed.Make lawn on the MHA plates.After owning make two vials by the help of borer one for aqueous extract of neem which served as control and second for ethanolic extract of neem.Now add 30 µl of both extracts into the vials.Incubate these plates at 37℃for 24 to 48 hours for the examination of zones.

Minimum inhibitory concentration method
For MIC take 9 tubes of 10 ml for each organism (E.coli, Pseudomonas, and Staph) having 1 ml MHA broth.Label the tubes as 0.25, 0.5, 1, 2, 4, 8, 16,32and control.From 1 ml discard respective microliters to maintain the volume except for the control.Now add 0.25, 0.5, 1, 2, 4, 8, 16, 32 µl of neem ethanolic extract by the help of jester and 1ml fresh culture in all 9 tubes so the control contains 1ml broth and 1ml culture.Properly cover the tubes and incubate the tubes at 37℃for 24-48 hours to check the non-turbid tubes.

Minimum bactericidal concentration method
Take MHA agar plates according to the nonturbid tubes.From the non-turbid tubes take 0.1 ml and streak on plates.Incubate the plates at 37C for 24 hours.Next day count the CFU/ml (30-300 colonies) to check the bactericidal activity of neem.Antibiotics sensitivity test Take three MHA plates and make lawn by using fresh cultures of (E.coli, Pseudomonas, and Staph).Place synthetic antibiotics discs of penicillin, Cephalosporin, Ampicillin, and Novobiocin on the MHA plates.Incubate the plates at 37C for 24 hours.Next day observed the zones.

Phytochemical analysis of neem leaves
To check the phytochemical analysis of neem leaves used different reagents.The compounds of neem are alkaloids, glycoside, flavonoid, triterpenes, tannins, and saponins.

Alkaloids
The test also called as Meyer's test.The extract of neem evaporated till dryness and the residue was heated with 2% HCl in a boiling water bath.After cooling and filtration add few drops of MEYER's reagent in the mixture.The turbidity or yellow precipitation shows the presence of alkaloids components.

Glycoside
To the extract add Glacial acetic acid, few drops of Ferric chloride and Concentrated Sulphuric acid.Reddish brown coloration at the junction of two layers and the bluish green color in the upper layer observed.Flavonoids 4 mg of extract solution treated with 1.5 ml of 50% methanol solution.In warmed solution add metal magnesium, and 5-6 drops of concentrated hydrochloric acid red color were observed for flavonoids.

Triterpenes
The extract mixed with 5 ml of chloroform, warmed at 80°C for 30 minutes.Add a few drops of concentrated sulphuric acid, mixed well red color formation appeared Tannins 0.5 ml of extract solution add 1 ml of water, 1-2 drops of ferric chloride solution.The bluish green color appears for tannins.

Saponins
In 2g powered sample of neem leaves were boiled a water bath containing 20 ml distilled water and filtered.10ml filtered solution mixed with 5ml of distilled water then add 2-3 drops of olive oil, shake well and observed for emulsion formation.

Results
The study was done to check and interpret the antibacterial activity of neem leaves to extract on human uro-pathogens like E. coli, Pseudomonas, and S. aureus by different methods including Agar well diffusion, antibiotic discs sensitivity test, Minimum inhibitory concentration, minimum bactericidal concentration.The ethanolic extract of neem show the zones of inhibition against Pseudomonas that is 23mm, E. coli that is 25mm, and for S. aureus is 26mm as shown in (Table 1).The result interprets that the maximum zone of inhibition of ethanolic extract of neem was found against S. aureus.The result of agar well diffusion compared with synthetic antibiotic disc diffusion against the same bacteria that cause urinary tract infection, the antibiotics used were cephalosporin, penicillin, ampicillin, and novobiocin.Presence of zones indicates that the neem extract has antibacterial activity.This activity is due to the presence of chemical and biological constituents like Alkaloids, Glycosides, Triterpenes, Saponins, Tannins, and flavonoids which can be determined by performing phytochemical screening of neem leaves which are mentioned below in (Table 2).Constituents like Tannins, Glycoside, Alkaloid, Saponins were found positive in the neem extract while Flavonoid and Triterpenes were found negative which indicates that Tannins, Glycoside, Alkaloid, Saponins have strong antibacterial activity.As per the Macrobroth dilution method performed, the results revealed that the neem extract inhibits the growth E. coli and Pseudomonas strains with the concentration 8ug/ml, 16 ug/ml, 32 ug/ml whereas the growth of S. aureus was inhibited 4, 8, 16, 32 but the MBC showed the colonies on MHA plates means having inhibitory effects not killing effect.

Minimum inhibitory concentrations (MIC) of neem extract
The minimum inhibitory concentration (MIC) of ethanol extracts of neem showed the highest inhibition for L. monocytogenes (0.1 mg/mL), S. aureus (strain 01) (0.1 mg/mL), S. aureus (strain 02) (0.1 mg/mL), and V. parahaemolyticus (0.1 mg/mL) and the lowest inhibition for Alcaligenesfaecalis, Aeromonashydrophila (4.0 mg/mL), and A. hydrophila (4.0 mg/mL).The MIC of chloroform extracts of neem showed similar inhibition for L. monocytogenes (4.0 mg/mL) and L. monocytogenes (5.0 mg/mL).However, ethanol extracts of neem showed higher inhibition for S. aureus (strain 01) (4.5 mg/mL) and S. aureus (strain 02) (4.5 mg/mL) and the lower inhibition for other microorganisms (6.5 mg/mL  glycosides, flavonoids, tannins, triterpenes, saponins.From this study, it may be concluded that the neem has an inhibitory effect not cidal effect against both grampositive and gram-negative organisms that caused UTI.In the future, the infections can be treat by using medicinal plant (neem) rather than the synthetic antibiotics which have side effects as well as these are more resistant to pathogens.So, we can be able to produce drugs which not only treat infections but also have no more side effects.
Conceived and designed the experiments: A Fatima & I Ibrahim, Performed the experiments: A Fatima & I Ibrahim, Analyzed the data: A Fatima & I Ibrahim,

Table 2 . Phytochemical screening of Neem leaves Name of organism Zone of inhibition Control
In the present study, found that the ethanolic extract of neem having these biologically active compounds like alkaloids, glycosides, tannins, flavonoids, triterpenes, saponins the antibacterial activity of neem extract may due to the presence of above-mentioned components [10].