Phytochemical Screening and Antibacterial Activity of Brillantaisia patula Leaf
Brillantaisia patula is a medicinal plant used for different ailments in Africa. Phytochemical constituents and antibacterial potential of the plant were investigated. In vitro antibacterial activity using agar-well diffusion method was carried out against Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 24212), Proteus hauseri (ATCC 13315), Pseudomonas aeruginosa ATCC 27853 and Escherichia coli (ATCC 38218). The phytochemistry of both methanol and ethanol extracts of Brillantaisia patula leaf revealed the presence of alkaloids, glycosides, terpenoids, steroids, flavonoids, tannins, steroids, flavonoids and saponins. Terpenoids and flavonoids were found in the methanolic extract but not in the ethanolic extract while steroid was found in the ethanolic extract but not in the methanolic extract. The ethanolic extract was active against all the five pathogenic bacteria while the methanolic extract inhibited all the test bacteria but Staphylococcus aureus. Ethanolic extract zones of inhibition ranged from 15.3±0.6 to 20.7±0.6 mm, whereas methanolic extract zones of inhibition ranged from 18.0±1.0 to 30.0±1.0 mm. The Minimum Inhibitory Concentration (MIC) of ethanolic extract ranged from 25 to 200 mg mL-1 while that of methanolic extract was from 50 to 100 mg mL-1. The Minimum Bactericidal Concentration (MBC) of methanolic extract ranged from 100 to 200 mg mL-1. The least MBC value (50 mg mL-1) of ethanolic extract was against Escherichia coli while the highest value (>200 mg mL-1) was against Proteus hauseri. Leaf of Brillantaisia patula could be a novel source of antibacterial agent (s) that might have broad spectrum activity.
to cite this article:
F. Faparusi, M.M. Bello-Akinosho, R.T. Oyede, A. Adewole, P.O. Bankole and F.F. Ali, 2012. Phytochemical Screening and Antibacterial Activity of Brillantaisia patula Leaf. Research Journal of Phytochemistry, 6: 9-16.
Received: October 20, 2011;
Accepted: November 30, 2011;
Published: February 13, 2012
Importance of plants to mans existence cannot be over emphasized. Plants
are used as sources of foods and medicines since the dawn of civilization (Sofowora,
1993). Many indigenous plants; trees, shrubs, herbs, twigs and leafy vegetable
are taken as food, spices or used for medicinal purposes in Nigeria (Nwaogu
et al., 2007). In Africa, where the larger percentage of the populace
lives in rural area, plants are used extensively in traditional health care
delivery in combating communicable and non-communicable diseases. According
to World Health Organisation (WHO), over 80% of the worlds population
depends on medicinal plants for their health care needs (Upadhyay
et al., 2011). Plant-derived medicines are relatively cheaper than
synthetic alternatives, offering profound therapeutic benefits and more affordable
treatment (Abubakar, 2009). Many of these plants are
readily available in rural areas, thus making traditional system of medicine
relatively cheaper than the orthodox medicine. The medicinal value of plants
depends on their chemical constituents that provide a definite physiological
action on the human body (Kumar et al., 2009).
These bioactive constituents are tannin, flavonoid, alkaloids, phenol compounds,
saponin, glycoside, anthraquinones etc. (Adegoke et al.,
2010; Duke, 1995; Kamba and Hassan,
2010; Ujowundu et al., 2010). The knowledge
of chemical constituents of plant is necessary for the discovery of therapeutic
agents of importance and their precursors. Many plant extracts are known to
have antimicrobial properties. In recent years, several studies have evaluated
antimicrobial activity of crude plant extracts (Adegoke
et al., 2010; Ayepola, 2009; Abubakar,
2009; Habtamu et al., 2010; Chhetri
et al., 2008; Rani et al., 2008; Kamba
and Hassan, 2010; Vinothkumar et al., 2010).
Brillantaisia patula belongs to the family Acanthaceae; it is a shrubby
plant of about 3 m height and can be found in Nigeria, Togo, west Cameroon and
across Uganda and Angola. The leaves are used to take care of yaws and rheumatism,
the decoction is taken to ease childbirth, menstrual pain and stomach ache.
It has been reported to have antiplasmodial and analgesic potentials (Makambila-Koubemba
et al., 2011; Mbatchi et al., 2006).
The present study was carried out to determine the chemical and antimicrobial
activity of leaf extracts of Brillantaisia patula.
MATERIALS AND METHODS
Plant material: The leaves of Brillantaisia patula were collected
during the months of December (2010)-February (2011) at Oke-Ola, Ilaro, Yewa-South
local government of Ogun State, Nigeria. The plant materials were authenticated
by Mr. P.O. Bankole, a botanist in Biology unit of Science Laboratory Technology
Department, Federal Polytechnic, Ilaro, Nigeria. The plant materials were cleaned
with distilled water, shade-dried at room temperature (27±2°C) and
pulverized using Kenwood electric blender (Kenwood Ltd., Harvant, United Kingdom).
The resulting powder was kept in air-tight container (27°C±2) until
Preparation of plant extracts: Crude solvent extraction of Brillantaisia
patula leaves was carried out using modified method of Omale
et al. (2010). The powdered leaves were soaked in ethanol (90%) and
methanol (90%) and filtered after 72 h. The filtrates were evaporated to dryness
using rotary evaporator.
Bacterial strains: The following gram-positive and gram-negative species were the test organisms used: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 24212), Proteus hauseri (ATCC 13315), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 38218) were used for in vitro antibacterial studies. All the stock cultures were obtained from the National Institute of Medical Research (NIMR) Yaba, Lagos State, Nigeria.
Phytochemical screening: Qualitative determination of phytochemical
constituents of the plant extracts was carried out using various methods. Iyengar
(1995) methods were followed for the determination of tannin, saponin and
reducing sugar. Methods of Siddiqui and Ali (1997) were
adopted for alkaloids, glyceroids, steroid, terpenoids, flavonoid and anthroquinones.
Phlobatanin was determined according to the method of Kumar
et al. (2009).
Purity test: Purity test was carried out on the crude extracts before use. Pour plate method and plate count agar were used for the test. The plates were incubated at 37°C for 24 h. The absence of growth was used as indicator of sterility of the extracts.
Determination of antimicrobial activity: The study was performed under
strict aseptic condition. The test organisms (bacteria) were sub-cultured into
nutrient broth and incubated at 37°C for 24 h. Normal saline was added to
adjust the turbidity to 0.5 McFarland standards which corresponds to 108
cells mL-1 (Habtamu et al., 2010;
NCCLS, 1990). Modified agar well diffusion method of
Habtamu et al. (2010) was adopted. The plates
were in triplicate and were left on the working bench for 30 min to allow for
diffusion before incubation at 37°C for 24 h (Esimone
et al., 1998). The diameter of the resulting zone of inhibition was
measured using transparent millimeter calibrated ruler.
Minimum Inhibitory Concentration (MIC): The methods described by El-Mahmood
et al. (2008) and Vinothkumar et al.,
2010 were used. The following concentrations; 200, 100, 50 and 25 mg mL-1
were prepared using two-fold serial dilution. The tubes were incubated at 37°C
for 24 h and observed for visible growth. The lowest concentration at which
no detectable bacterial growth occured was considered as Minimum Inhibitory
Minimum Bactericidal Concentration (MBC): The minimum bactericidal concentration
of the extract was determined by selecting tubes that showed no growth during
MIC determination. A loopful was taken from the test tubes and inoculated on
sterile Muller Hinton agar by streak plate method in triplicates. The plates
were incubated at 37°C for 24 h. The lowest concentration of the extracts
that showed no colony growth on the solid medium was regarded as minimum bactericidal
concentration (Mann et al., 2008).
Statistical analysis: The results of zones of inhibition were presented
as mean of replicates and Standard Deviation (SD).
Alkaloids, glycosides, terpenoids, steroids, flavonoids, tannins and saponins were present in either or both extracts. Alkaloids, glycosides, tannins and saponins were present in both extracts. Terpenoids and flavonoid were present in the methanolic extract but not in ethanolic extract. Steroid was present in ethanolic extract, not in methanolic extract. Phlabotanins and reducing sugar were absent in both extracts (Table 1).
The susceptibility of the test organisms to the crude extracts on the basis
of zones of inhibition varied according to organisms and extraction solvents.
Methanolic and ethanolic extracts showed varying degrees of activity against
the test organisms. Brillantaisia patula leaf ethanolic was active against
the five test organisms; Enterococcus faecalis, Proteus hauseri, Escherichia
coli, Staphylococcus aureus and Pseudomonas aeruginosa. The methanolic
extract showed activity against all the test organisms except Staphylococcus
aureus. Standard antibiotic (pefloxacin) only showed activity against
Enterococcus faecalis, Staphylococcus aureus and Pseudomonas aeruginosa.
|| Phytochemical screening of Brillantaisia patula leaf
|+: Present; -: Absent
|| Antimicrobial activity of Brillantaisia patula leaf
|E: Ethanolic; M: Methanolic; PFX: Pefloxacin; R: Resistant
|| Minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) of ethanolic extract of Brillantaisia patula leaf
|+: Growth; -: No growth
The zones of inhibition of extracts ranged from 15.3±0.6 and 31.3±1.2
mm. Ethanolic extract showed the highest and least zones of inhibition against
Escherichia coli and Proteus hauseri, respectively. As shown in
Table 2, extraction solvents (control) were not active against
the test organisms. The zones of inhibition of methanolic extract ranged from
18.0±1.0 and 30.0±1.0 mm.
Ethanolic extract inhibited Enterococcus faecalis, Proteus hauseri, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at minimum concentration of 50, 200, 25, 50 and 50 mg mL-1, respectively. Ethanolic extracts demonstrated bactericidal activity against Enterococcus faecalis and Pseudomonas aeruginosa at minimum concentration of 100 mg mL-1. Ethanolic extract also showed MBC against Escherichia coli and Proteus hauseri at concentrations of 50 and >200 mg mL-1, respectively (Table 3).
||Minimum inhibitory concentration of (MIC) and minimum bactericidal
concentration (MBC) of methanolic extract of Brillantaisia patula leaf
|+: Growth; -: No growth
Methanolic extract of B. patula inhibited Enterococcus faecalis, Proteus hauseri and Pseudomonas aeruginosa at a minimum concentration of 100 mg mL-1 and Escherichia coli was inhibited at minimum concentration of 50 mg mL-1 as shown in Table 4. Methanolic extract demonstrated bactericidal activity against Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa at minimum concentration of 100 mg mL-1 (Table 4). Methanolic extract also showed MBC at a concentration of 200 mg mL-1 against Proteus hauseri (Table 4).
The study revealed the antibacterial potential of Brillantaisia patula
and its phytochemical constituents. The presence of notable phytochemicals;
alkaloids, glycosides, terpenoids, steroids, flavonoids, tannins and saponins
in the plant leaf might have been responsible for its uses in traditional health
care. These bioactive constituents (secondary metabolites) have been associated
with antimicrobial activities in mammalian cells (Mishra
et al., 2009; Nweze et al., 2004;
Sofowora, 1993). Several phenolic compounds like tannins
are potent inhibitors of many hydrolytic enzymes such as proteolytic macerating
enzymes used by plants pathogens (Kamba and Hassan, 2010).
Non-toxic glycoside can also be hydrolysed to release phenolic compounds that
are toxic to microbial pathogens. These bioactive compounds exert antimicrobial
activity through different mechanisms. Tannins are known to form irreversible
complexes with proline-rich protein (Shimada, 2006),
resulting in the inhibition of microbial cell protein synthesis. Plants with
tannins as their main components are astringent in nature and are used for treating
intestinal disorder such as diarrhoea and dysentery (Dharmananda,
2003). Tannins also have anticancer activity and can be used in cancer prevention
(Okuda and Ito, 2011), thus Brillantaisia patula
has the potential as a source of vital bioactive compounds for the management
of cancer. Cardiac glycosides have good potentials in the field of cancer therapy
and treatment of heart failure (Prassas and Diamandis, 2008;
Schoner and Schein-Bobis, 2007) are naturally cardioactive
drugs used in the treatment heart failure and cardiac arrhythmia (Agbafor
and Nwachukwu, 2011). The extracting solvents played important role in the
phytochemical constituents of the extracts as revealed by the variation in the
phytochemical constituents of methanolic extract and ethanolic extract of the
same plant. This variation could be due to difference in the extraction ability
of the solvents. This finding was in agreement with the work of Egharevba
et al. (2010).
The antibacterial assays showed that extracts of Brillantaisia patula
leaf have antipathogenic bacteria potential. Both extracts showed significant
antibacterial activity, but ethanolic extract was more active based on the number
of test bacteria inhibited. The two extracts showed varying zones of inhibition.
Habtamu et al. (2010) also reported difference
in antibacterial activity with different extraction solvents. The antibacterial
activity of the plant could be due to phytochemicals identified in the extracts.
Various works have reported antimicrobial activity of different medicinal plants
(Adegoke et al., 2010; Ayepola,
2009; Chhetri et al., 2008; Abubakar,
2009; Habtamu et al., 2010; Rani
et al., 2008; Kamba and Hassan, 2010). The
crude extracts compared favourably with the standard antibiotic (pefloxacin)
based on the zones of inhibition but crude extract were better in terms of number
of test bacteria inhibited. Inability of methanolic extract to inhibit Staphylococcus
aureus might be due the concentration of extract used for the antibacterial
The MIC and MBC assays showed the highest MIC and MBC values against Proteus hauseri. The bacteria was less susceptible to the crude extracts, the organism might also be carrying gene that could partially hydrolyse part of the active constituents of the extracts. Escherichia coli was the most susceptible with least MIC and MBC values, Brillantaisia patula could be a potential source of antibacterial agents for the management of enteric disorder. The in vitro antibacterial activity of Brillantaisia patula might not be enough to justify its potential, there is need for the in vivo antibacterial activity to be evaluated.
The methanolic and ethanolic extracts of Brillantaisia patula contained a good number of bioactive chemical constituents including alkaloids, glycosides, terpenoids, steroids, flavonoids, tannins and saponins. Extraction solvents played important role in the phytochemical constituents of the extracts. Based on the number of phytochemicals identified in the extracts, methanol was a better solvent. Ethanolic extract was more active against all the test organisms. Leaf extract of Brillantaisia patula could be a novel source of antibacterial agent (s) that might have broad spectrum activity.
Adegoke, A.A., P.A. Iberi, D.A. Akinpelu, O.A. Aiyegoro and C.I. Mboto, 2010.
Studies on phytochemical screening and antimicrobial potentials of Phyllanthus amarus
against multiple antibiotic resistant bacteria. Int. J. Applied Res. in Nat. Prod., 3: 6-12.Direct Link |
Agbafor, K.N. and N. Nwachukwu, 2011.
Phytochemical analysis and antioxidant property of leaf extracts of Vitex doniana
and Mucuna pruriens
. Biochem. Res. Int.,CrossRef |
Ayepola, O.O., 2009.
Evaluation of the antimicrobial activity of root and leaf extracts of Terminalia glaucescens
. Adv. Nat. Applied Sci., 3: 188-191.Direct Link |
Chhetri, H.P., N.S. Yogol, J. Sherchan, K.C. Anupa, S. Mansoor and P. Thapa, 2008.
Phytochemical and antimicrobial evaluations of some medicinal plants of Nepal. Katumandu Univ. J. Sci., Eng. Technol., 1: 49-54.CrossRef | Direct Link |
Dharmananda, S., 2003.
Gallnuts and the uses of tannins in Chinese medicine. Institute for Traditional Medicine, Portland, Oregon. http://www.itmonline.org/arts/gallnuts.htm.
Duke, A.T., 1995.
Handbook of Medicinal Herbs. 3rd Edn., CRS Press, London, Pages: 220
Egharevba, H.O., O.E. Ocheme, G. Ugbabe and M.S. Abdullahi, 2010.
Phytochemical screening and antimicrobial studies of methanol, ethyl acetate and hexane extracts of Vitex doniana
, sweet (stem, bark and leaf). Nat. Sci., 8: 177-185.Direct Link |
Abubakar, E.M.M., 2009.
Antibacterial efficacy of stem bark extracts of Mangifera indica
against some bacteria associated with respiratory tract infections. Scient. Res. Essay, 4: 1031-1037.Direct Link |
El-Mahmood, A.M., J.H. Doughari and N. Ladan, 2008.
Antimicrobial screening of stem bark extracts of Vitellaria paradixa
against some enteric pathogenic microorganisms. Afr. J. Pharm., 2: 89-94.Direct Link |
Esimone, C.O., M.U. Adujwym and J.M. Okonta, 1998.
Preliminary antimicrobial screening of the ethanolic extract from the Lichen Usnea subfloridans
(L). J. Pharm. Res. Dev., 3: 99-101.
Habtamu, Y., T. Eguale, A. Wubete and T. Sori, 2010. In vitro
antimicrobial activity of selected Ethiopian medicinal plants against some bacteria of veterinary importance. Afr. J. Microbiol. Res., 4: 1230-1234.Direct Link |
Iyengar, M.A., 1995.
Study of Crude Drugs. 8th Edn., Manipal Power Press, Manipal, India, Pages: 2
Kamba, A.S. and L.G. Hassan, 2010.
Phytochemical screening and antimicrobial activities of Euphorbia balsamifera
leaves, stems and root againstsome pathogenic microorganisms. Afr. J. Pharm. Pharmacol., 4: 645-652.Direct Link |
Kumar, A., G. Rajput, V.K. Dhatwalia and G. Srivastav, 2009.
Phytocontent screening of mucuna seeds and exploit in opposition topathogenic microbes. J. Biol. Environ. Sci., 3: 71-76.Direct Link |
Makambila-Koubemba, M.C., B. Mbatchi, D. Ardid, A. Gelot and C. Henrion et al
Pharmacological studies of ten medicinal plants used for analgesic purposes in congo brazzaville. Int. J. Pharmacol., 7: 608-615.CrossRef |
Mann, A., A. Banso and L.C. Clifford, 2008.
An antifungal property of crude plant extracts from Anogeissus leiocarpus
and Terminalia avicennioides
. Tanzania J. Health Res., 10: 34-38.CrossRef | PubMed | Direct Link |
Mbatchi, S.F., B. Mbatchi, J.T. Banzouzi, T. Bansimba and G.F.N. Ntandou et al
., 2006. In vitro
antiplasmodial activity of 18 plants used in Congo Brazzaville traditional medicine. J. Ethnopharmacol., 104: 168-174.CrossRef | PubMed | Direct Link |
Mishra, A.K., A. Mishra, H.K. Kehri, B. Sharma and A.K. Pandey, 2009.
Inhibitory activity of Indian spice plant Cinnamomum zeylanicum extracts against Alternaria solani
and Curvularia lunata
, the pathogenic dematiaceous moulds. Ann. Clin. Microbiol. Antimicrob., Vol. 8,CrossRef | Direct Link |
Methods for Antimicrobial Susceptibility Testing. In: Manual of Clinical Microbiology, Balows, A. (Ed.). 5th Edn., America Society for Microbiology, Washington, DC., USA., pp: 1125-1150
Nwaogu, L.A., C.S. Alisi, C.O. Ibegbulem and C.U. Igwe, 2007.
Phytochemical and antimicrobial activity of ethanolic extract of Landolphia owariensis
leaf. Afr. J. Biotechnol., 6: 890-893.Direct Link |
Nweze, L.A., J.L. Okafor and O. Nwoku, 2004.
Methanolic extracts of Treme guineenes
(Schumm and thom) and Morinda lucida
Benth used in Nigeria traditional herbal medicinal practice. Biol. Res., 2: 33-48.
Okuda, T. and H. Ito, 2011.
Tannins of constant structure in medicinal and food plants-hydrolyzable tannins and polyphenols related to tannins. Molecules, 16: 2191-2217.CrossRef | Direct Link |
Omale, J., S.C. Enemuor and E.E. Hussaaini, 2010.
Antimicrobial and antioxidant activity of Saba florida
(benth) extracts. J. Biosci. Tech., 1: 180-186.Direct Link |
Prassas, I. and E.P. Diamandis, 2008.
Novel therapeutic application of cardiac glycosides. Nat. Rev. Drug Discovery, 7: 926-935.CrossRef | Direct Link |
Rani, I., S. Akhund and H. Abro, 2008.
Antimicrobial potentials of seed extract of Raphanus sativus
. Pak. J. Bot., 40: 1793-1798.Direct Link |
Shimada, T., 2006.
Salivary proteins as a defense against dietary tannins. J. Chem. Ecol., 32: 1149-1163.Direct Link |
Siddiqui, A.A. and M. Ali, 1997.
Practical Pharmaceutical Chemistry. 1st Edn., CBS Publishers and Distributors, New Delhi, pp: 126-131
Sofowora, A., 1993.
Medicinal Plants and Traditional Medicine in Africa. 2nd Edn., Spectrum Books Ltd., Ibadan, Nigeria, ISBN-13: 9789782462190, Pages: 289
Ujowundu, C.O., O.E. Okafor, N.C. Agha, L.A. Nwaogu, K.O. Igwe and C.U. Igwe, 2010.
Phytochemical and chemical composition of Combretum zenkeri
leaves. J. Med. Plants Res., 40: 965-968.Direct Link |
Upadhyay, H.C., D.C. Saini and S.K. Srivastava, 2011.
Phytochemical analysis of Ammannia multiflora
. Res. J. Phytochem., 5: 170-176.CrossRef |
Vinothkumar, P., A. Sivaraj, K.S.Z. Ahmed, P. Sivamani, K. Devi and B. Senthilkumar, 2010.
Evaluation of antibacterial activities of Andrographis paniculata
leaf extract against gram positive and gram negative species by in vitro
methods. J. Pharm. Res., 3: 1513-1515.Direct Link |
Schoner, W. and G. Scheiner-Bobis, 2007.
Endogenous and exogenous cardiac glycosides: Their roles in hypertension, salt metabolism and cell growth. Am. J. Physiol. Cell Physiol., 293: C509-C536.CrossRef | Direct Link |