Phytochemical Screening and Antimicrobial Activity of Three Medicinal Plants against Urinary Tract Infection Pathogens

Urinary tract infections are a global health issue. Although antibiotics exist, most people for fear of stigmatization often prefer medicinal treatments. Three plants Acanthus montanus, Aspilia africana and Desmodium velutinum were investigated for their antimicrobial activity against human urinary tract infection pathogens and also screened for phytochemicals. The phytochemical screening and quantification, characterisation of isolates, antimicrobial screening and minimum inhibitory concentration were all done using standard techniques. Replicate readings were then subjected to analysis of variance. The results of the phytochemical screening and quantification of the plants showed the presence alkaloids (2.40 – 3.12%), glycosides (3.50 – 4.20%), saponins (3.02 -6.27%), tannins (0.17 – 0.47%), flavonoids (9.22 – 11.42%), polyphenol (9.72 – 9.90%) and Original Research Article Ebana et al.; AJMAH, 1(2): 1-7, 2016; Article no.AJMAH.29460 2 reducing compounds (7.409.18 mg %) in studied plants. Analysis of the replicate readings showed significance (p < 0.05). The results of the microbial characterization showed that isolates were Esherichia coli, Staphylococcus species and Pseudomonas aeruginosa. The antimicrobial sensitivity assay showed that the isolates responded differently to the test plants extracts. Staphylococcus species and E. coli were more sensitive to the test extracts than P. aeruginosa. The least inhibition was 11.50 mm while the highest was 14.00 mm. Consistently, all the studied plants showed very good inhibition at a minimum inhibitory concentration of 200 mg. The findings in this study confirms their use in the treatment of urinary tract pathogen and the need for further studies aimed at determining the bioactive compounds in these plants.


INTRODUCTION
Urinary tract infections (UTIs) remains one of the most common infections globally with no less than half of all women having atleast one episode of UTIs during their life time [1,2].They affect any part of the urinary tract which could be the kidney, ureter, bladder and urethra.The causes of UTIs includes sexual intercourse with infected persons, poor hygiene, holding urine longer than necessary, using diaphragm singly or with spermicides or condoms, underlying kidney stones, diabetes, loss of estrogen and catheter [3].Most common pathogens remain Esherichia coli while others include Staphylococcus species, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella and so on [1,3].With a global estimated 150 million cases of UTIs annually, there are concerns that UTIs are on the increase and one reason for this is the emergence of antibiotics resistance amongst UTIs pathogens [2].As a result of this, alternative strategies have been sought and used in the treatment of UTIs and amongst them is the use of medicinal plants [1,4].
A study in Bangladesh has shown that people with these infections for fear of social stigmatization prefer to visit traditional medicinal healer even when health facilities may be available or within reach.Furthermore, the study identified a number of medicinal plants that are used to treat a variety of UTIs such as cloudy urine, leucorrhoea, menstrual pains, lower abdominal pains, burning sensation, presence of blood or pus or semen in urine, and difficulties in passing urination and frequent or lack of urination, bleeding of the penis and so on.Interestingly, different tribes had different medicinal plant for their treatment of various UTIs ailments and they include plant (s) in the families of acanthaceae, adiantaceae, amarathaceae, apiaceae, apocynaceae, asterceae, bigoniaceae, convolvulaceae, costaceae, cucurbitaceaee, euphorbiaceae, fabaceae, lauraceae, malvaceae, melastomataceae, menispermaceae, nymphaeaceae, rhamnaceae and rutaceae [4].
The use of medicinal plants is not just the custom of the distant past.It is projected that about 70-90% of the world's population still relies on raw herbs and unrefined extracts [5].In southern Nigeria, a number of medicinal plants are used by locals to treat urinary tract infection and they include Acanthus montanus, Desmodium velutinum and Aspilia africana.
Aspilia africana is a regular weed that is native to forest zones of Nigeria and other West African countries [6].The flowers and leaves of this plant have several therapeutic uses such as enhanced wound healing and stoppage of bleeding from cuts.The methanolic extract of the leaves is reported to cure malaria and respiratory ailments [7].Furthermore, the concoctions of the leaves are used to cure eye problems, ringworm and dysentery [8].Although they are not popular as food, A. africana have been shown to be rich in vitamins such as ascorbic acid, niacin, thiamin and riboflavin, and minerals such as calcium, nitrogen, magnesium, potassium, phosphorus, zinc, nickel, lead and boron.They have also been to shown to be rich in phytochemical such as alkaloids, saponins, flavonoids, tannins and phenols [8].
Acanthus montanus commonly called Bear's breeches is another plant that has a place in folk medicine where it is used to manage body aches and pains, cough and even threatened abortion [9].Phytochemical evaluation of the plant shows that it is rich in steroids, glycosides, alkaloids and tannins amongst others.The roots extracts have been used in the treatment of urinogenital infection, urethral pains and even endometriosis [10].Using Swiss Mice model, a study has shown that it has potentials as an herbal solution to spermatogenesis malfunction [11].
Desmodium velutinum is a perennial plant that is erect or semi-erect growing up to about 3 meters in length.Like the former plants, it is widely distributed in the tropics and sub-tropics [12].Traditionally, the extracts have been used in the treatment of aches, pains, diarrhoea and haematuria when used with hot peppers [12].Furthermore, it is used as a diuretic, laxative, treatment of cough, and fever amongst others.Studies have shown that it is rich in phytochemicals such as tannins, resins, glycosides, saponins and flavonoids [12,13].
Given the medicinal potentials and the dearth of information on these plants despite their uses in the management of urinary tract health infections, the aim of this study was therefore to examine the phytochemicals and antimicrobial activity of these plants extracts against urinary tract infection pathogens.

Source and Identification of Plants under Study
Freshly

Collection of Test Bacteria and Characterization
Urinary tract infection pathogens were collected from the University of Uyo Teaching Hospital from patients presenting with signs and symptoms of UTIs.The isolates were reidentified using standard microbiological procedures and they included Esherichia coli, Staphylococcus species and Pseudomonas aeruginosa.They were appropriately preserved in slants until required for use [15][16][17].

Sample Preparation
The fresh leaves were hand-picked into a clean basin and made into a powder as previously described [18][19].Briefly, the picked leaves were rinse with sterile distilled water and allowed to air dry by spreading it in a clean stainless tray.After which, the leaves were oven dried using an electric oven maintained at 60°C for 3 hours.Following drying, the resulting leaves were then pulverized using a mortar and pestle to produce a powder.This was repeated for all the leaves and the powders were then stored in sample bottles until required.

Preparation of Plants Extracts
This was done as previously described [18][19].Both ethanolic and aqueous extracts were prepared using 95% ethanol and distilled water, respectively.Briefly, the ethanolic extracts of all the plants were prepared by weighing out and dissolving 50 g of the powdered plants in a sterile beaker containing exactly 1000 ml of the ethanol and distilled water.The mixture was stirred vigorously for a few minutes and allowed to stand for 72 hours wrapped with aluminum foil.After which, it was filtered using a Whatman filter paper and the solvent heated in a water bath to evaporate completely to produce a slurry.These were then repeated for all the plants and aqueous extract.These slurries were then stored in McCartney bottles and kept at 4°C until needed.

Antimicrobial Sensitivity Testing
Antimicrobial sensitivity testing was carried out on the extracts using the discs diffusion method previously reported [18][19][20].Briefly, a cork borer was used to cut filter papers into tiny disks of 5mm in diameter.The disks were then sterilized using an autoclave set at 121°C for 15 minutes.Colonies of each test isolates were then subcultured on nutrient broth and incubated at 37°C for 6 hours.They were then inoculated on freshly prepared Mueller-Hinton agar plates.The sterilized filter paper disks were soaked in the respective test extracts and then placed on the plates aseptically.The plates were incubated at 37°C for 24 hours.After incubation, the zones of inhibition were determined in triplicates and on separate days.The mean values were then determined and recorded.

Minimum Inhibitory Concentration (MIC)
The MIC of the plants extracts were determined using pour plate technique.Different concentration of 20 mg, 100 mg and 200 mg were prepared by weighing out 0.10 g, 0.50 g and 1.00 g of each of the extracts and dissolving them in 5 ml of water separately.Sterilized 5 mm discs were soaked in the various concentrations for 10 minutes and allowed to air dry.These were then incubated with the pour plates of the test organisms at 37°C for 24 hours.The plates were then examined for the lowest inhibitory concentration for each of the plant extracts and test organisms.

Statistical Analysis
The replicate data obtained in this study were managed and analysed using Statistical package for social science (SPSS) version 21.The results were then presented as mean ± sd (standard deviation).Probability values less than 0.05 were considered significant at 95% level of significance.

RESULTS
The results of the present study are presented in the tables below.Table 1 gives the plant part used in this study including their scientific, local and family names.Table 2     Nigeria.Phytochemical screening of the leaves showed an abundance of phytochemicals.All the leaves are found to contain saponins, glycosides, saponins, flavonoids, tannins, polyphenols and reducing compounds.The most abundant phytochemical was polyphenol that was present atleast in excess in all the leaves extracts.An earlier study showed the presence of tannins, alkaloids, saponins, steroids, hydrogen cyanide, carbohydrate, flavonoids, reducing sugars and terpenoids in D. velutinum collected in Enugu, South East Nigeria and this conforms to the findings in our study [21].Alkaloids, steroids and proteins were absent in the leaves of D. velutinum harvested from Anambra State but it was found to have glycosides, flavonoids, tannins, resins and saponin [22].
The phytochemicals recorded in our study for A. africana were similar to that found in another study [8].The presence of alkaloids, saponins, flavonoids, tannins and phenols is similar to the findings in our study.However, on quantification, the most abundant phytochemical was alkaloid in their study while it was flavonoid in our study.Acanthus montanus have been shown to be rich in phytochemicals such as alkaloids, flavonoids, phenols, saponins, steroids and tannins [23].In this study, on quantification, the most abundant phytochemical was steroids followed by flavonoids.This was contrary to our findings which gave flavonoids as the most abundant followed by polyphenol.
The result of the antimicrobial sensitivity of the leaves of A. montanus, A. africana and D. velutinum shows that the extracts have antibacterial activity on urinary tract isolates.The highest zone of inhibition seen on the isolates was 14.00 mm using ethanolic extract of A. africana with Staphylococcus species.All extracts whether aqueous or ethanolic showed inhibitory effects on the isolates used in this study.Zones of 27.50 mm, 21.00 mm, 22.50 mm, 25.00 mm, 19.00 mm and 18.50 mm were reported against E. coli, S. aureus, S. pneumoniae, P. mirabilis, P. aeruginosa and K. pneumonia with A. montanus.These zones were far greater than that reported in our study [23].Our findings are more agreeable to those reported earlier [10].In their study, they reported zones of 15.00 mm and 19.30 mm for 100 mg/ml of the aqueous extract of A. montanus against P. aeruginosa and S. aureus, respectively.The MIC for all the medicinal plants under study was consistent at 200 mg.

CONCLUSION
The present study shows the presence of phytochemicals and the antimicrobial potential of the medicinal plants against urinary tract isolates.The findings in this study confirm their use as medicinal plants against urinary tract infection pathogens and thus the need for further exploitation.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

COMPETING INTERESTS
Authors have declared that no competing interests exist.

Table 1 . Plant used in the study
Polyphenol was the most abundant in all the plant extracts.Table3gives the result of the phytochemical quantification and it shows that A. africana had the highest amount of reducing compounds, alkaloids and saponins.However, A. montanus had the highest amount of polyphenols while D. velutinum had the highest amount of glycosides, tannins and flavonoids.Replicate readings of the phytochemical expressed as mean±sd were significant (p < 0.05).Table4shows the antimicrobial sensitivity results of the aqueous and ethanolic extracts of the studied plants.None of the isolates were resistant to the plant extracts used.The highest zone of inhibition of 14.00 mm was seen with the

Table 3 . Quantitative estimation of crude phytochemicals
*Represent significant Mean±SD that were significant across each column (p < 0.05)