Studies on The Antibacterial Effect of The Ethanolic

J o u r n a l o f M e d i c a l B i o m e d i c a l a n d A p p l i e d S c i e n c e s

J Med Biomed App Sci 8 (2), 343–351 (2020) ISSN (O) 2349-0748

Studies on The Antibacterial Effect of The EthanolicExtracts of Cnidoscolus aconitifolius (Miller) (HospitalToo Far), Piliostigma thonningii (Schum) (Camels Foot)And Lantana camara (Linn) (Lantana)

Nyam M.A⋆1, Abdullahi U.I.1, Atsen E.1, Itelima J.U.1

Department of Plant Science & Biotechnology, University of Jos

DOI: 10.15520/jmbas.v8i2.212

Accepted 5 February 2020; Received 25 January 2020; Publish Online 8 February 2020

Reviewed By: Dr.Daniel V.

ABSTRACTStudies were carried out at National Veterinary Research Institute (NVRI) VomJos, Plateau State on antibacterial activities of ethanolic leaf and stem extracts ofCnidoscolus aconitifolius, Piliostigma thonningii and Lantana camara to verify claimsby locals of their medicinal properties. Standard cold maceration qualitative biochem-ical analysis and agar well diffusion methods were used. All extracts were effectiveagainst gram positive and gram negative bacteria with the exception of Shigel lady sen-teriae which showed resistance against four extracts but was highly positive against theextracts of Piliostigma thonningii.A positive control Ciprofloxacin was tested againstall clinical isolates producing a broad spectrum of activity, positive zones of inhibitionwere screened for minimum inhibitory concentration (MIC) and minimum bacterici-dal concentration (MBC). Results of biochemical screening of the test plants revealedthe presence of alkaloids, tannins, flavonoids, carbohydrates, steroids, anthraquinones,and cardiac glycosides, with saponins only present in Piliostigma thonningii stem andLantana camara leaf extracts. Sensitivity test was carried out against two gram pos-itive bacteria. Bacillus subtilis and Staphylococcus aureus, and three gram negativebacteria Escherichia coli, Salmonella typhi and Shigel lady senteriae. From the re-sults of these findings, the activity of the extracts might be concentration-dependentbecause all the best activities were seen at the highest concentration (200mg/ml)and therefore, work on higher concentrations and purification of the crude extractsis recommended to achieve considerable antibacterial activity. The results providedscientific backing to the use of these plant extracts by the locals in the treatment ofconditions usually associated with the organisms tested.Key words: C. aconitifolius–P. thonningii–L. camara–Bacteria–Leaf–Stem–Extracts

1 INTRODUCTIONOver time, there have been increased and concerted ef-forts towards the adoption and integration of traditionalmedicine and medicinal practices referred to as complemen-tary or/and alternative medicine in both developing anddeveloped countries in their respective health systems [1].This was after a study by WHO demonstrated that 80%of the world’s population depends on medicinal plants fortheir primary healthcare [2, 3].

⋆ Corresponding author.

Chemo therapeutics which involves the extraction anddevelopment of drugs from plants and their derivatives hasbeen carried out over the years in order to counter the effectof many diseases that affect us. This goes to say that specificplants called medicinal plants are of great importance in themanufacture of drugs since the chemical components of theplants are used for the production of these drugs [4].

Rural and urban populations in some parts of West Africause certain plant species for therapeutic and dietary pur-poses. Among the plants traditionally used by people is acultivated plant belonging to the Euphorbiaceae family, aleguminous plant belonging to the Caesalpiniaceae and an

10.15520/jmbas.v8i2.212

344 Nyam M.A et al.

evergreen shrub belonging to the family Verbaneaceae.The family Eurphorbiaceae includes Cnidoscolus aconi-

ifolius (Miller) which is also frequently consumed. C. aconi-tifolius, known as tree spinach (English), efoiyanaipaja orefoJerusalem (Yoruba) [5]. It is also referred to as ‘Hospitaltoo far’ [6]. It is commonly found growing in the Westernpart of Nigeria. It is an ornamental evergreen, drought de-ciduous shrub of 3 to 5m tall [7]. The crop originated asa domesticated leafy green vegetable in the Maya regionof Guatemala, Belize and Southeast Mexico during pre-Cambrian period (Ross-Ibara and Mollina-Cruz, 2002). Itis cultivated in domestic gardens rather than in Agricul-tural fields and as such can be used throughout the year. Itis a widely distributed annual plant, ranging from temper-ate to tropical zones, and has a long history of use as bothmedicinal and edible plant [8]. It has certain antibacterialproperties as well as a contraceptive effect [9]. It has beenobserved in use as diuretic, circulation and lactation stimu-lants and has also been recommended for diabetes, obesity,acne, kidney stones and eye problems [10].

Piliostigma thonningii leguminous plant belonging to thefamily Caesalpiniaceae. The tree is perennial in nature andits petals are white to pinkish coloured produced betweenNovember and April. It is locally known as Mukuura inMbeere, wild bauhinia, camels foot, Embu county in Kenyahas been used and reported for different and varied medic-inal purposes. It has been reported that in many Africancountries, various parts of the plant (roots, barks, seed andfruit) are used to treat wounds, ulcers, gastric, and heartpain, gingivitis and as an antipyretic [11, 12].

Piliostigma thonningii (Schum) is also found growingabundantly as a wild uncultivated small tree in many partsof Nigeria such as Minna, Plateau, Zaria, Ilorin, Lagos andAbeokuta [13]. It is a leguminous plant belonging to thefamily Caesalpiniaceae, a family that comprises trees andshrubs. Attention has been drawn to this plant becauseof several claims on its enormous applications as an ethnomedicinal plant used to cure several diseases including thecommon malaria, dysentery, fever infectious respiratory ail-ments, hydropsy sterility, rachitis and skin diseases. [14].

Studies independently shows phytochemical evaluation,antibacterial activity [15] and anti-malaria activity [16].

Lantana camara is a low erect, rugged hairy, evergreenshrub (Verbanaceae) native to tropical America. Known byseveral common names viz, black sage, cuasquito, angel lip,flowered sage, shrub verbena, white sage and wild sage allover the world, it is a significant weed of which there aresome 650 varieties in over 60 countries or island groups.L. camara has several uses, mainly as herbal medicine andin some areas as firewood and mulch. It is also used forthe treatment of cancers, chicken pox, measles, asthma, ul-cers, swellings, eczema, tumors, high blood pressure, biliousfevers, catarrhal infections, tetanus, rheumatism, malariaand atoxy of abdominal viscera [17]. Extracts from the lan-tana leaves exhibit antimicrobial, insecticidal and nematici-dal activity and also contain verbascoside, which possessesantimicrobial, immunosuppressive and antitumor activities.It also has certain antibacterial properties [17].

In view of the reputed efficacies of these plants, thepresent study aimed at investigating the phyto chemi-cals constituents and antibacterial activities of these plantsagainst some selected bacteria in an attempt to determinewhether their traditional use as medicine are supported byactual pharmacological effect or merely based on folklore.

1.1 Statement of ProblemThe tendency of populations in developing countries tofavour traditional plants is mainly due to the inaccessibilityof modern medical care, as well as economic and culturalfactors [18].

Infectious diseases continue to be the major causes ofmulti drug resistant strains of bacteria and a lack of new an-tibiotic classes in the drug development, alternative strate-gies are needed to manage bacterial infections [19] hence,the increasing interest in the search for effective antibacte-rial agents with wide spectrum of activities which are cheapand safer.

1.2 JustificationA range of medicinal plants with antimicrobial propertieshave been widely used by the traditional healers, studiesof some have shown, the effectiveness of certain traditionalmedicine in treating infectious diseases [20]. However mostof the research work carried out are not authenticated thusthe need for this work to authenticate the traditional claimsof the antibacterial potential of C. aconitifolius, P. thon-ningii, and L. camara.

1.3 Aim And ObjectivesThe broad aim of this research is to establish a scientificbase for the traditional uses of the extracts of Cnidoscolusaconitifolius, Piliostigma thonningii and Lantana camarafor treating bacterial infections.

While the specific objectives are to:1. determine the biochemical constituents of C. aconiti-

folius, P. thonningi, and L. camara leaves and stem extractsusing standard procedures.

2. determine the antibacterial susceptibility effect of se-lected pathogenic microbes to the C. aconitifolius, P. thon-ningii, and L. camaraleaf and stem extracts.

3. determine the minimum inhibitory concentration(MIC) and minimum bactericidal concentration (MBC) ofthe leaf and stem ethanolic extracts of C. aconitifolius, P.thonningii, and L. camara.

4. determine the effects of the leaf and stem extracts of C.aconitifolius, P. thonningii, and L. camara as antibacterialagents.

2 MATERIALS AND METHODS2.1 Study LocationThis research work was carried out at the Microbiology lab-oratory of the Dermatophilosis Research Centre of National

Journal of Medical Biomedical and Applied Sciences , Vol 8 Iss 2, 343–351 (2020)

Studies on The Antibacterial Effect of The Ethanolic Extracts of Cnidoscolusaconitifolius (Miller) (Hospital Too Far), Piliostigma thonningii (Schum) (Camels Foot)

And Lantana camara (Linn) (Lantana) 345

Veterinary Research Institute (NVRI), Vom Plateau State.Vom is quite a rocky village in Jos South Local Govern-ment Area of Plateau State and situated at an elevation ofabout 1,238 meters or 4,062 feet high above sea level. Thenearest towns are Bukuru and Jos, 12.8km and 24km to thenorth east respectively. Largely because of its altitude andconstant wind, Vom has a remarkably cool climate.

2.2 Plant CollectionFresh leaves and stems of the selected plants were collectedfrom the Nursery of Federal College of Forestry Jos. Theplants were identified by a taxonomist at the Herbariumof the Federal College of Forestry Jos. The samples werecarefully taken to the Pharmacognosy laboratory of the de-partment of Pharmaceutical Sciences University of Jos, andwashed with deionized water to remove the surface contam-inants. They were then dried at ambient temperature in thelaboratory to avoid heat destruction of the active compo-nents of the samples. After drying the samples were thenground into powdered form with sterile laboratory pestleand mortar to increase the surface area. 100g each of theground samples were kept in airtight polythene bags.

2.3 Plant ExtractionThe pulverized samples (100g each) were macerated in 70%ethanol and subjected to shaking for 48 hours using a me-chanical shaker. [21]. After soaking, it was filtered usingWhat-man no. 1 filter paper and the filtrates were cen-trifuged and the supernatant poured out. The residues werethen poured into a conical flask and evaporated to almostdryness (this is to prevent the extracts from sticking to thewalls of the flask and to enable complete recovery of all theextracts). After the samples were successfully evaporated,the weight of the extracts was determined and stored forsubsequent analysis.

2.4 Sources of Bacterial IsolatesThe test organisms used were two grams-positive bacte-ria namely Bacillus subtilis and Staphylococcus aureus andthree gram – negative bacteria namely Escherichia coli,Salmonella typhi and Shigelladysenteriae were used for theexperiment. The bacterial isolates were obtained from thebacteriology unit of the Central Diagnostic Laboratory ofNational Veterinary Research Institute (NVRI) Vom Jos,Plateau State.

2.5 Preparation of The Test OrganinsmsThe isolates were sub-cultured onto nutrient broth and re-identified using recommended biochemical test.

2.6 Standardization of InoculumsFive colonies of each bacterial isolates were inoculated intonutrient broth (Oxoid, UK) incubated at 370C for 24 hours.Turbidity produced was adjusted to match 0.5 McFarlandturbidity standards using a Nephelometer.

2.7 Antibacterial Susceptibility TestNutrient agar (NA) was used for the evaluation of antibacte-rial activity. Prepared sterile plates of NA already solidifiedand 24 hour-old standardized cultures of the different bac-teria were introduced using streaking method. A sterile corkborer of 4mm diameter was used to make wells according tothe different concentrations. About 0.1ml of reconstitutedserial diluted extract equivalent to 2mg of the extract wasdropped into each approximate labeled well and the pos-itive control ciprofloxacin (40mg.ml) was introduced intothe wells and a negative control was also prepared whichhas only the media and the standardized organisms. Theinoculated plates were left on the table for 1 hour to allowthe extracts to diffuse into the agar. The plates were thenincubated aerobically at 370C for 24 hours. Zones of inhibi-tion produced after 24 hours of incubation were measuredin millimeters [22].

2.8 Determination of Minimum Inhibitory Concen-tration (Mic)

The Minimum Inhibitory Concentration was carried out todetermine the lowest concentration that would show no vis-ible growth. Serial dilution of the extracts was carried outagain according to the different concentrations. 13g of nu-trient broth powder was diluted in 1 litre of distilled water.The solution was well mixed and sterilized in an autoclave at1210C for 15 minutes. 10ml of the prepared nutrient brothwas dispensed into sterile test tubes according to the con-centrations, 0.5ml of the extract and a loopful of the stan-dardized organisms were added to the broth shaken andincubated for 24 hours at 370C. A positive control was alsoplaced for comparison which contained neither of the ex-tract nor the organism. The test tubes were observed forturbidity after 24 hours and recorded.

2.9 Determination of Minimum Bactericidal Con-centration (Mbc)

The tubes from the MIC that showed no visible growthwere plated out again by streaking a loopful of the contentfrom the MIC over the surface of an already prepared andsolidified sterile nutrient agar plate which was incubated for24 hours at 370 C and observed.

3 RESULTS3.1 Results of The Biochemical Analysis of The

Test PlantsSource: Laboratory work, 2018.

KeyCa = Cnidoscolus aconitifoliusPt = Piliostigma thonningiiLc = Lantana camara+++ = Present in appreciable amount++ = Present in moderate amount+ = Present in minute amount


346 Nyam M.A et al.

Table 1. Biochemical constituents present in the plant extractsof Cnidoscolus aconitifolius, Piliostigma thonningii and Lantanacamara

Con-stituents

Leaf(Ca)

Stem(Ca)

Leaf(Pt)

Stem(Pt)

Leaf(Lc)

Stem(Lc)

Alkaloids +++ ++ +++ - ++ +++Saponins - - - ++ +++ -Tannins +++ + +++ ++ - +++Flavonoids +++ +++ +++ + +++ +++Carbohy-drates

+ ++ +++ ++ + +++

Steroids ++ - +++ + - +Terpenes +++ - - - - -An-thraquinones

- - +++ - - -

Cardiacglycosides

++ + ++ - +++ -

- = Not detectedThe biochemical analysis of the leaf extract of Cnidosco-

lus aconitifolius revealed the presence of alkaloids, tannins,flavonoids, carbohydrates, steroids, terpenes and cardiacglycosides while stem extracts showed the presence of al-kaloids, tannins, flavonoids, carbohydrates and cardiac gly-cosides. P. thonningii leaf extract showed the presence ofalkaloids, tannins, flavonoids, carbohydrates, steroids, an-thraquinones, and cardiac glycosides while the stem extractshowed the presence of saponins, tannins, flavonoids, carbo-hydrates and steroids. The biochemistry of Lantana camaraleaf extract revealed the presence of alkaloids, saponins,flavonoids, carbohydrates, and cardiac glycosides while thestem extract showed the presence of alkaloids, tannins,flavonoids, carbohydrates and steroids.

3.2 Antibacterial Susceptibility of The Plant Ex-tracts

Table 2(a) Zones of inhibition (mm) of the antibacterial activity of the leaf extract of C. aconitifolius.

Concentra-tions(mg/ml)

Testorganisms

200 100 50 25 12.5 +vecon-trol

Bacillussubtilis

15 12 10 9 0 26.0

Staphylococ-cusaureus

16 12 8 2 0 28.0

Escherichiacoli

17 13 9 5 0 26.0

Salmonellatyphi

21 20 18 15 6 28.5

Shigelladysen-teriae

0 0 0 0 0 22.0

Source:Laboratorywork, 2018.

The leaf extract showed the highest activity againstSalmonella typhi, followed by Escherichia coli, Staphylococ-cus aureus and the Bacillus subtilis. The extract showed noactivity against Shigel lady senteriae at 200mg/ml. C. aconi-tifolius can be used in the treatment of diseases caused byS. typhi, E. coli.

Table 2(b) Zones of inhibition (mm) of the antibacterial activity of the stem extract of C. aconitifolius

Test organisms 200 100 50 25 12.5 +vecontrol

Bacillus subtilis 18 13 9 7 4 26Staphylococcusaureus

19 18 15 12 0 28

Escherichia coli 14 9 6 3 0 26Salmonella typhi 20 16 16 13 6 28.5Shigelladysente-riae

0 0 0 0 0 22

Source: Laboratory work, 2018.The extract showed the highest activity against

Salmonella typhi followed by S. aureus, B. subtilis and thenE. coli. The extract showed no activity against Shigella-dysenteriae at 200mg/ml.

Table 3(a) Zones of Inhibition(mm) of the Antibacterial Activity of the Leaf Extract of P. thonningii

Concentrations(mg/ml)Test organisms 200 100 50 25 12.5 +ve

controlBacillus subtilis 20 17 14 10 0 26Staphylococcusaureus

19 17 17 15 0 28

Escherichia coli 9 6 3 0 0 26Salmonella typhi 18 14 10 8 8 28.5Shigelladysenteriae 21 18 16 96 22

The extract showed the highest activity against Shigel-ladysenteriae followed by Bacillus subtilis, Staphylococcusaureus, Salmonella typhi, with the least activity against Es-cherichia coli.

Table 3(b) Zones of Inhibition(mm) of the Antibacterial Activity of the Stem Extract of P. thonningii.



16 11 10 8 0 28

Escherichia coli 12 12 8 4 0 26Salmonella typhi 0 0 0 0 0 28.5Shigelladysenteriae 15 8 8 6 2 22Source: Laboratorywork, 2018.

The extract shows the highest activity against S. aureus,followed by S. dysenteriae, B. subtilis and the E. coli, theextract showed no activity against S. typhiat 200mg/ml.




Table 4(a) Zones of Inhibition(mm) of the Antibacterial Activity of the Leaf Extract of L.camara



15 12 12 8 0 28

Escherichia coli 10 10 8 6 0 26Salmonella typhi 4 2.5 2 2 0 28.5Shigelladysenteriae 0 0 0 0 0 22Source: Laboratorywork, 2018.

The extract showed the highest activity against B. sub-tilis, followed by S. aureus, E. coli and then S. typhi. Theextract showed no activity against Shigelladysenteriae at200mg/ml.

Table 4(b) Zones of Inhibition (mm) of the Antibacterial Activity of the StemExtract of L. camara.



15 12 6 0 0 28

Escherichia coli 12 8 5 5 0 26Salmonella typhi 20 13 10 10 6 28.5Shigelladysenteriae 0 0 0 0 0 22

The extract showed the highest activity against S. ty-phi, followed by S. aureus, B. subtilis and then E. coli.The extract showed no activity against, Shigelladysenteri-aeat 200mg/ml.

Table 5(a) Minimum Inhibitory Concentration (MIC) of the Leaf Extract of C.aconitifolius

Concentrations(mg/ml)Test organisms 200 100 50 25 12.5 MICBacillus subtilis - - + + + 100Staphylococcus aureus - - - + + 50Escherichia coli - - + + + 100Salmonella typhi - - - - + 25Shigelladysenteriae 0 0 0 0 0 0

Key- = No turbidity+ = Presence of turbidityTable 5a showed the MIC of the leaf extract of C. aconi-

tifolius against the test organisms. Negative signs (-) showsclarity while the positive signs (+) shows turbidity.

- = No turbidity+ = Presence of turbidityThe above table shows the MIC of the stem extract of C.

aconitifolius against the test organisms. Negative signs (-)shows clarity while the positive signs (+) shows turbidity.

Table 5(b) Minimum Inhibitory Concentration (MIC) of the Stem Extract of C.aconitifolius

Concentrations(mg/ml)Test organisms 200 100 50 25 12.5 MICBacillus subtilis - - - + + 50Staphylococcus aureus - - - + + 50Escherichia coli - - + + + 100Salmonella typhi - - - + + 50Shigelladysenteriae 0 0 0 0 0 0

Table 6(a) Minimum Inhibitory Concentration (MIC) of the Leaf Extract of P.thonningii.

Test organisms 200 100 50 25 12.5 MICBacillus subtilis - - + + + 100Staphylococcusaureus

- - - - + 25

Escherichia coli - + + + + 200Salmonella typhi - - - - + 25Shigelladysenteriae - - - - + 25

Table 6a shows the MIC of the leaf extract of P. thon-ningii against the test organisms. The negative signs (-)shows clarity while the positive signs (+) shows turbidity.0

Table 6(b) Minimum Inhibitory Concentration (MIC) of the Stem Extract of P. thonningii.

Concentrations(mg/ml)Test organisms 200 100 50 25 12.5 MICBacillus subtilis - - + + + 100Staphylococcus aureus - - + + + 50Escherichia coli - - + + + 100Salmonella typhi + + + + + 0Shigelladysenteriae - - - + + 50

Tables 6a and 6b show the MIC of the stem extract ofP. thonningii against the test organisms. The negative signs(-) shows clarity while the positive signs shows turbidity.

Table 7(a) Minimum Inhibitory Concentration (MIC) of the Leaf Extract of L. camara.

Concentrations(mg/ml)Test organisms 200 100 50 25 12.5 MICBacillus subtilis - - + + + 100Staphylococcus aureus - - + + + 50Escherichia coli - + + + + 200Salmonella typhi - + + + + 200Shigelladysenteriae 0 0 0 0 0 0

Table 7a shows the MIC of the leave extract of L. camaraagainst the test organisms. Negative signs (-) shows claritywhile the positive signs shows turbidity.

The above table shows the MIC of the stem extract of L.camara against the test organisms. Negative signs (-) showsclarity while the positive signs shows turbidity.

Key – No growth, + Presence of growth.Table 8a shows the MBC of the leaf extract of C. aconiti-

folius against the test organisms, negative signs (-) indicates


348 Nyam M.A et al.Table 7(b) Minimum Inhibitory Concentration (MIC) of

the Stem Extract of L.camara.

Concentrations(mg/ml)Test organisms 200 100 50 25 12.5 MICBacillus subtilis - - + + + 100Staphylococcus aureus - - + + + 100Escherichia coli - + + + + 200Salmonella typhi - - - + + 50Shigelladysenteriae 0 0 0 0 0 0

Table 8(a) Minimum Bactericidal Concentration (MBC) of the Leaf Extract of C. aconitifolius

Test organisms 200 100 50 25 12.5 MBCBacillus subtilis - - + + + 100Staphylococcusaureus

- - + + + 100

Escherichia coli - - + + + 100Salmonella typhi - - - + + 50Shigelladysenteriae 0 0 0 0 0 0

the absence of growth (bactericidal) while the positive signs(+) indicates the presence of growth.

Table 8(b) Minimum Bactericidal Concentration (MBC) of the Stem Extract of C. aconitifolius.

Concentrations(mg/ml)Test organisms 200 100 50 25 12.5 MBCBacillus subtilis - - + + + 100Staphylococcusaureus

- - + + + 50

Escherichia coli - - + + + 100Salmonella typhi - - - + + 50Shigelladysenteriae 0 0 0 0 0 0

Key- No growth+ Presence of growthTable 8b indicates the MBC of the stem extract of C.

aconitifolius against the test organisms. Negative signs (-) indicates the absence of growth (bactericidal) while thepositive signs (+) indicates the presence of growth.

Table 9(a) Minimum Bactericidal Concentration (MBC) of the Leaf Extract of P. thonningii

Concentrations(mg/ml)Test organisms 200 100 50 25 12.5 MBCBacillus subtilis - - + + + 100Staphylococcusaureus

- - - + + 50

Escherichia coli - + + + + 200Salmonella typhi - - - + + 50Shigelladysenteriae - - - - + 25

Table 9a shows the MBC of the leaf extract of P. thon-ningii against the test organisms, negative signs (-) showsthe absence of growth (bactericidal) while the positive signs(+) shows the presence of growth.

Table 9(b) Minimum Bactericidal Concentration (MBC) of the Stem Extract of P.thonningii

Concentra-tions(mg/ml)

Testorganisms

200 100 50 25 12.5 MBC

Bacillussubtilis

- - + + + 100

Staphylococ-cusaureus

- - + + + 100

Escherichiacoli

- - - + + 200

Salmonellatyphi

- - - + + 0

Shigella-dysenteriae

- - - + + 50

The above table shows the MBC of the stem extract ofP. thonningii against the test organisms, negative signs (-)shows the absence of growth (bactericidal) while the positivesigns (+) shows the presence of growth.

Table 10(a) Minimum, Bactericidal Concentration (MBC) of the Leaf Extract of L. camara.

Concentration(mg/ml).

Testorganisms

200 100 50 25 12.5 MBC

B. subtilis - - + + + 100S. aureus - - + + + 100E .coli - + + + + 200S. typhi - + + + + 200S. dysen-teriae

0 0 0 0 0 0

The above table shows the MBC of the leaf extract of L.camara against the test organisms. Negative signs (-) showsthe absence of growth (bactericidal) while positive signs (+)shows the presence of growth.

Table 10(b) Minimum Bactericidal Concentration (MBC) of the Stem Extract of L. camara.

Concentration(mg/ml).Test organisms 200 100 50 25 12.5 MBCB. subtilis - - + + + 100S. aureus - - + + + 100E. coli - + + + + 200S. typhi - - + + + 100S. dysenteriae 0 0 0 0 0 0

Table 10b shows the MBC of the stem extract of L. ca-mara against the test organisms. Negative signs (-) showsthe absence of growth (bactericidal) while the positive signs(+) shows the presence of growth.




Figure 1. Photograph of Cnidoscolus aconitifolius plant, Pil-iostigma thonningii plant and Lantana camara plant

Figure 2. Photograph of thezones of inhibition on different or-ganisms

Figure 3. Photograph of MIC determination

Figure 4. Photograph of MBC determination

4 DISCUSSIONBiochemical analysis carried out on all the test plantethanolic extracts showed the presence of some bioactivecompounds in the plants. Cnidoscolusaconitifolius showedthe presence of alkaloids, tannins, flavonoids, carbohy-drates, steroids, terpenes, and cardiac glycosides.This find-ing is in consonance with previous reports by [23] and thepresence of flavonoids is in complete contrast to the re-port by [24]. The result of the biochemical screening on Pil-iostigma thonningii showed that out of the nine active ingre-dients screened for, seven were present in the plant namely,alkaloids, tannins, flavonoids, carbohydrates, steroids, an-thraquinones, cardiac glycosides.

Lantana camara showed the presence of alkaloids,saponins, tannins, flavonoids, carbohydrates, steroids, and

cardiac glycosides. The presence of these phytochemicalcompounds may be responsible for its antibacterial activ-ity as confirmed by [25], owing to the fact that these plantsdemonstrated considerable antibacterial activity as seen inthe results of the sensitivity test.

The presence of zones of inhibition on these plants showedthat, the plants extracts possesses antibacterial activity onboth Gram Positive and Gram Negative bacteria. Althoughthe zones of inhibition were lower than that exhibited bythe standard drug Ciprofloxacin, this could be due to thefact that the plant extracts are crude and contain otherconstituents that do not possess antibacterial properties.

The zones of inhibition of the growths of the microor-ganisms employed in the work were compared among thedifferent plants tested. Bacillus subtilis showed the highestactivity at the highest concentration of the leaf extract ofPiliostigma thonningii followed by the leaf extract of Lan-tana camara, stem extract of Cnidoscolus aconitifolius, leafextract of Cnidoscolus aconitifolius, stem extract of Lantanacamara and then the stem extract of Piliostigma thonningii.All the plant extracts showed no activity against Bacillussubtilis at the lowest concentration with the exception of thestem extract of Cnidoscolus aconitifolius which producedzone of inhibition at the lowest concentration.

Staphylococcus aureus produced the highest activity atthe highest concentration of the stem extract of Cnidoscolusaconitifolius which produced an equal zone on the leaf ex-tract of Piliostigma thonningii followed by the leaf extractof Cnidoscolus aconitifolius which also produced equal zonewith the stem extract of Piliostigma thonningii and boththe leaf and stem of Lantana camara produced equal zonesof inhibition. All the plant extracts showed no activity atthe lowest concentration.

Escherichia coli produced the highest activity at the high-est concentration of the leaf extract of Cnidoscolus aconi-tifolius followed by the stem extract of Cnidoscolus aconi-tifolius, the stem extract of Piliostigma thonningiiand thestem extract of Lantana camara produced equal activitywhile the leaf extract of Piliostigma thonningii producedthe least activity at the highest concentration, all the plantsextracts showed no activity against E. coli at the lowest con-centration 12.5mg/ml.

Salmonella typhi produced the highest activity at thehighest concentration of the leaf extract of Cnidoscolusaconitifolius followed by the stem extract of Cnidoscolusaconitifolius and stem extract of Lantana camara whichproduced the same zone of inhibition, then the leaf extractof Piliostigma thonningii. All the activities were significantagainst S. typhibut surprisingly, the stem extract of Pil-iostigma thonningii produced no activity against this or-ganism and the leaf extract of Lantana camaraproduced aninsignificant inhibition. This organism showed activity evenat the least concentrations of the leaf extract of Cnidosco-lusaconitifolius, stem extract of C. aconitifolius, leaf extractof P. thonningiiand the stem extract of L. camara respec-tively.

Shigelladysenteriae has proven to be resistant, produc-ing no activity against leaf and stem extracts of Cnidosco-lusaconitifoliusand Lantana camara, but produced a highly


350 Nyam M.A et al.

significant activity on the leaf extract of Piliostigma thon-ningii which compete favourably with the positive control.The organism also produced a significant activity on thestem extract of Piliostigma thonningii. In both the leaf andstem extract of P. thonningii, activity was observed even atthe lowest concentrations.

Furthermore, the minimum inhibitory concentration(MIC) was determined against the five clinical isolates.The MIC of Bacillus subtilis against C. aconitifolius leafand stem extracts were 100mg/ml and 50mg/ml, that ofP.thonningii leaf and stem extracts were 100mg/ml and100mg/ml and Lantana camara leaf and stem extracts were100mg/ml,and100mg/ml respectively. The MIC of S. aureusagainst the plant extracts of C.aconitifolius were 50mg/mland 50mg/ml, that of P. thonningii were 25mg/ml and50mg/mland L. camara were 50mg/ml, and 100mg/ml re-spectively. The MIC of E. coli against the plant extractsof C. aconitifolius were 100mg/ml and 100mg/ml, that ofP. thonningi were 200mg/ml and 100mg/ml and L.camarawere 100mg/ml and 100mg/ml respectively. The MIC of thestem extract of P. thonningii against S. typhi was not deter-mined because it does not show any positive inhibition. TheMIC of S. dysenteriae was only determined for the plant ex-tracts of P. thonningii where the leaf extract showed MICof 25mg/ml and the stem extract was 50mg/ml, all otherextracts showed no positive activity against S. dysenteriae.

The Minimum bactericidal concentration (MBC) was de-termined against the test organisms. The MBC of B. subtilisagainst the plant extracts of C. aconitifoliuswere 100mg/mland 100mg/ml that of P.thonningiiwere 100mg/ml and100mg/ml and L. camara was found at 100mg/ml, and100mg/ml respectively. The MBC of S. aureus against thetest plants were 100mg/ml, 50mg/ml, 50mg/ml, 100mg/ml,100mg/ml, and 100mg/ml respectively. The MBC of E.coli against the test plants were 100mg/ml, 100mg/ml,200mg/ml, 200mg/ml, 200mg/ml and 200mg/ml respec-tively. The MBC of S. typhi against the test plants were50mg/ml, 50mg/ml, 50mg/ml, 200mg/ml and 100mg/ml re-spectively. The MBC of dysenteriae was only determinedagainst the leaf and stem extracts of P. thonningii whichwere 25mg/ml and 50mg/ml respectively.

Generally, all the extracts showed significant difference asantibacterial agents against B. subtilis, S. aureus, E. coli, S.typhi and S. dysenteriae. These findings agree with earlierstudies by [24, 26].

Various literatures indicate that an extract has bacteri-cidal activity when the MBC value is the same or generallynot more than four-fold higher than the MIC; and bacterio-static when more than four-fold or many-fold higher thanthe MIC. The closer the MIC to its MBC, the more bac-tericidal it is. From this context this study demonstratesthat C. aconitifolius, P. thonningii and L. camara ethanolicextracts possess bactericidal compounds against one of thetest organisms or the other.

5 CONCLUSIONThe result of this investigation indicated that C. aconiti-folius, P. thonningiiand L. camara extracts contained somesubstantial amounts of biochemicals that exhibited good an-tibacterial activities and this might have supported the var-ious folk applications of different extracts of the test plantsto cure ailments from time immemorial. The mechanism ofresistance of bacteria to antibiotics does not confer resis-tance to the compound present in the test plants.

The growth of bacterial resistance to antibiotics is athreat to the world population with an increasing reoc-currence of infectious diseases due to the emergence ofmulti drug resistant bacteria that hinder chemotherapy [27].Therefore, in accordance with the established standards, theethanolic extracts of the test plants showed activity againstboth Gram-positive and Gram-negative bacteria, therebysuggesting that it could serve as a broad spectrum antibac-terial agent and paving way for further investigation to iden-tify the active compounds responsible for the plants’ biolog-ical activities.

6 RECOMMENDATIONIt is recommended that purification of the crude extracts bedone to ascertain the particular compound that is involvedin the antibacterial activity and further research should bein vivo to ascertain its potency in vivo. Further work shouldalso be carried out on the test plants at a higher concentra-tion to determine its susceptibility.

REFERENCES[1] National policy on traditional medicine and regulation of

herbal medicines. World Health Organization. 2005;.[2] Mothana RA, Abdo SA, Hasson S, Althawab FM. An-

timicrobial Antioxidant and cytotoxic Activities and phyto-chemical screening of some Yemeni Medicinal Plants. EvidBased complement Altern Medicine. 2008;7(3):323–353.

[3] Gupta VK, Sharma SK. Invitro Antioxidant Activities ofAqueous Extract of Ficusbangalensis Linn. Root Interna-tionalJournal ofbiologicalchemistry. 2010;4(3):134–140.

[4] Houghton PJ, Raman A. Laboratory handbook for fraction-ationof Naturalextract. London: Chapman and Hall; 0199.

[5] Olayinka AO, Onoruvwe O, Lot TY; 1992.[6] Yakubu MT, Akanji MA, Oladiji AT, Olatinwo AWO, Ades-

okan AA, Yakubu MO, et al. Effects of Cnidoscolusaconiti-folius(miller) IM Johnson leaf extract on reproductive hor-mones of female rats. Iran Journal ReprodMed. 2008;6:149–159.

[7] Kuti JO, Torres ES. Potential nutritional and health ben-efits of tree spinach. vol. 520. JJ, editor. Arlington, VA:Progress inNew Crops. ASHS Press; 1996.

[8] Nebel S, Heinrich M. Ta Chorta: A comparative ethnobotan-ical linguistic study of wild food plants in a Graecanic areain Calabria Southern Italy. Econs Bot. 2009;63(1):78–92.

[9] Dong CX, Hayashi K, Lee J, Hayashi T. Characterizationof Structures and Antiviral Effects of Polysaccharides fromPortulacaoleracea L chem. Pharmaceutical. 2010;58(4):507–510.

[10] Yuan W, Lis, By SO, Zhang Z, Wang P, Zhang W, et al.Flavonoid, Coumarins and triterpenes from the aerial parts




of Cnidoscolustexanus. Planta Med. 2007;73:1304–1308.[11] Silva NCC, Fernandes J. Biological Properties of Medicinal

Plants: A review of their antimicrobial activity. JournalVenom Nim Incl Trop Dis. 2010;16(3):402–413.

[12] Maroyi A. Traditional use of medicinal plants in south-central Zimbabwe: review and perspectives. Journal of Eth-nobiology Ethnomedicine. 2013;9:31–31.

[13] Keay RWJ. Trees of Nigeria. New York: Oxford UniversityPress; 0196.

[14] Tira-Picos V, Nogueira JM, Gbolade AA. Comparativeanalysis of leaf essential oil constituents of Piliostigmath-onningiiand Piliostigmareticulatum. International JournalGreen Pharmacology. 2010;4:67–70.

[15] Tor-Anyin TA, Anyam JV. Phytochemical evaluation andantibacterial activity: A comparison of various extracts fromsome Nigerian trees peak. Journal of Medicinal PlantsRe-search. 2013;1(2):13–18.

[16] Kwaji A, Bassi PU, Aoil M, Nneji CM, Ademowo G. Pre-liminary studies on Piliostigmathonningiischum leaf extract.Journal of Microbiology. 2010;.

[17] Craig WL; 2006. Available from: http://www.iso-/orc.[18] Abbiw O. Mis-uses andabuses inself -medicationwith medic-

inal plants. The case of Erthrophelum in Ghana. vol. 720. M,LJG, BXM, RJM, editors. Netherlands: Kluwer AcademicPublisher; 1996.

[19] Digiandomenico A, Keller AE, Gao C, Rainey GJ, WarrenerP, Camara MM, et al.; 2014.

[20] Ibewuike JC, Ogungbamila FO, Ogundaini AO, Okeke IN,Bohlin L. Anti-inflammatory and antibacterial activitiesof C-methyl flavonoids from Piliostigmathonningii. Ltd.1996;61(2):186–190.

[21] Ewansiha JU, Garba SA, Mawak JD, Oyewole O. Antimi-crobial Activity of Cymbopogoncitratus (lemon grass) andits Phytochemical Properties. Front Sci. 2012;2(6):214–220.

[22] Abayomi S; 0200.[23] Peixoto S, Tjs, Castro V, Saraiva AM, ADM, Pisciottano

TEA, et al. Phytochemical screening and antibacterial ac-tivity of four Cnidoscolus species (Eurphorbiaceae) againststandard strains and clinical isolates. Journal of MedicinalPlants Research. 2012;6(21):3742–3748.

[24] Awoyinka OA, Balogun IO, Ogunnowo AA. Phytochemi-cal Screeining and in vitro Bioactivity of Cnidoscolusaconi-tifolius (Euphorbiaceae). Journalof Medicinal Plants Re-search. 2007;1:63–65.

[25] Robinson N; 2006.[26] Asuzu IA, Gray AI, Waterman PG. The Antihelmintic

Activities of D-3-Methylchiroinositol from Piliostigmathon-ningii Stem Bark. Fitoerapia. 1999;70:77–79.

[27] Aqil F, Khan M, Ahmad I. Effect of Certain Bioactive PlantExtracts on Clinical Isolates of B-lactamase Producing Me-thicillin Resistant Staphylococcus aureus. Journalof BasicMicrobiology. 2005;45:106–114.


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Studies on The Antibacterial Effect of The Ethanolic