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Phcog J | Dec 2012 | Vol 4 | Issue 34 1
O R I G I N A L A R T I C L EP H C O G J
ABSTRACT
Introduction: Dalbergiella nyasae (Baker f.) belongs to the family Papilionaceae, small to medium sized herbal
plant well known for its medicinal properties in the treatment of gastrointestinal infections including diarrhoea.
This study aimed to determine the in vitro antimicrobial activity of crude extracts and fractions from leaves and
root bark of D. nyasae against selected bacteria and yeast of gastrointestinal relevance. Methods: Fourteen
extracts from leaves and root bark of D. nyasae have been screened for the antimicrobial activity against three
bacteria species including Gram negative E. coli and P. aeruginosa, Gram positive S. aureus and one yeast species
C. albicans using agar well diffusion, micro broth dilution and Total Activity (TA) analysis methods. Crude extracts
have been qualitatively screened for the presence of phytoconstituents and HPLC fingerprint profiles determined.
Results: Excellent antibacterial and antifungal activity against Gram positive S. aureus and one yeast species
C. albicans respectively were observed in n-butanol fraction of leaves extract. In root bark, best antibacterial activity
against S. aureus was observed in ethanol extract while against P. aeruginosa was observed in acetone extract.
For E. coli, best antibacterial activity was recorded in ethanol extract. Phytochemical analysis demonstrated the
presence of alkaloids, flavonoids, saponins and terpenoids. The HPLC fingerprint profiles of leaves extract recorded
one major peak whereas root bark extract recorded two major peaks. Conclusion: This investigation established
a good support for use of D. nyasae plant by traditional healers in Malawi as herbal medicine for gastrointestinal
infections and a base for development of novel potent drugs and phytomedicine.
Keywords: Agar well diffusion, In vitro antimicrobial activity, micro broth dilution, phytomedicine, Total Activity
(TA) analysis.
Extraction, characterization and pharmacological
evaluation of leaves and root bark of Dalbergiella
nyasae (Baker f.)
Frank Ngonda,*¹ Zakalia Magombo,³ Placid Mpeketula¹ and Jonas Mwatseteza²
¹Department of Biological Sciences, Chancellor College, University of Malawi, P.O. Box 280, Zomba, Malawi
²Department of Chemistry, Chancellor College, University of Malawi, P.O. Box 280, Zomba, Malawi
³National Herbarium and Botanic Gardens of Malawi, P.O. Box 528, Zomba, Malawi
Submission Date: 27-9-2012; Review completed: 10-11-2012; Accepted Date: 4-12-2012
INTRODUCTION
Globally, over 10 million under five children die every
year mostly from bacterial and fungal related infections
(diarrhoea, pneumonia, meningitis, etc) and about 90%
of these children come from the developing countries.[1]
And the major cause of this morbidity and mortality in
the developing countries includes among others poor
availability of interventions and spread and emergency
of antimicrobial resistance.
Multiple drug resistance has become a real problem in
pharmacotherapeutics due to an increasing number of
diseases exhibiting various levels of drug resistance.[2]
Furthermore, the development of synthetic drugs has
slowed down as a result of drug resistance.[3]
Conse-
quently, this has created a new renewed interest in the
search for new drugs in order to combat resistance.
*Corresponding author.
Frank Ngonda
Department of Biological Sciences, Chancellor College
University of Malawi, P.O. Box 280, Zomba, Malawi
E-mail: ngondafb@yahoo.com
Placid357@yahoo.com
jfmwatseteza@cc.ac.mw
zlkmagombo@hotmail.com
DOI:
3. Frank Ngonda, et al.: Extraction, characterization and pharmacological evaluation of leaves and root bark of Dalbergiella nyasae (Baker f.)
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Herbal medications and phytochemical screening of
various plant species for medicinal leads are now receiv-
ing much attention. Some of the herbal medicines that
are being considered as a source of new antibacterial
drugs have been time-tested for thousands of years
and are comparatively safe for both human use and the
environment.[4]
The plant genetic resource base in Africa
has an enormous potential to provide diverse chemical,
enzymes and genes that has remained unexploited at
industrial scale for production and design of new phar-
maceutical products.[5]
The plant Dalbergiella nyasae (vernacular name: mlem-
bera or mkanganjovu), is native to Malawi Mozambique,
Zambia, Zimbabwe and Tanzania, and belongs to the
family of Papilionaceae. It is found in deciduous wood-
land and thickets, usually small to medium in size with
leaves crowded near the ends of branches and inparipin-
nate with 6–9 pairs of oval leaflets and have a terminal
leaflet.[6]
Despite the widespread use of Dalbergiella nyasae as tra-
ditional medicine in Malawi for treatment of infectious
diseases, neither its phytochemistry nor pharmacologi-
cal effects has been evaluated and reported on the effi-
cacy of purported medicinal preparations. In Malawi,
the predominant medicinal system in use is that of
traditional medicine, especially in the rural areas where
there is limited Government health services such as
drug shortage, health personal and insufficient number
of hospitals and up to 80% of the population relies on
herbal plants as a source of primary health care.[7]
How-
ever, most of the indigenous information on herbal
remedies is passed on from generation to another as
folk tales without documentation.[8]
Therefore, the pur-
pose of this validation study was to identify the active
principles in the plant extracts and to investigate the
in vitro antimicrobial effect of the crude extract and
fractions of leaves and root bark of Dalbergiella nyasae
used by traditional health practitioners in the treatment
of gastrointestinal infections such as diarrhoea and
dysentery.
MATERIAL AND METHODS
Plant material
The medicinal plant (Dalbergiella nyasae) was collected
from Chingale area, in Zomba district (Southern Region
of Malawi) and identified by Mr I.H. Patel at Malawi
National Herbarium and Botanic Gardens with voucher
specimen number 69951 (Salubeni, Tawakali and Mjojo
5840 (MAL) Chingale, Zomba 15°24ʹS 35°11ʹE). The
root bark and leaves were separately shade dried, finely
powdered using a blender and kept in airtight polyethyl-
ene bags at room temperature in the dark until used.
Microorganisms
Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus
and Candida albicans were obtained from the Department
of Microbiology, University of Malawi. The test strains
were maintained on nutrient agar slant at 4°C and sub-
cultured on nutrient broth for 24 hrs prior to testing.
Direct extraction and solvent-solvent extraction
of plant material
One gram of finely powdered sample was extracted three
times with 10 ml of solvents (acetone, ethanol and dis-
tilled water) with vigorous shaking. The extracts were
decanted after centrifuging at 5300 × g for 10 minutes
and solvents removed at room temperature.[9]
For solvent-solvent extraction, 100 grams of finely pow-
dered plant material was extracted with 1 litre of acetone
(technical grade-Merck) in macerating bottle. The bottle
was shaken for 1 hr, 6 hr and 24 hr on shaking machine
and extracts decanted. Six grams of the extract collected
was fractionated using solvents of varying polarities
(dichloromethane, hexane, butanol, ethyl acetate, aque-
ous methanol and water). All the solvents were removed
under reduced pressure using rotary evaporator at 45ºC
and dried under room temperature.
Determination of antimicrobial activity
The agar well diffusion method[10]
was used to assay the
extracts for antimicrobial activity. 0.2 ml of 1 in 100 dilu-
tions of bacterial and fungal cultures (2.5 × 105
cfu ml–
¹)
was added to 20 ml of the melted and cooled Mueller Hin-
ton Agar (MHA) and Sabourand Dextrose Agar (SDA)
respectively. The contents were mixed by gentle swirling
movements before being poured into sterile petri dishes.
After solidification of agar, wells (6 mm) was bored in
each plate. 100 µl of each extract dissolved in acetone was
poured into appropriately labelled well.
Diameter of zones of inhibitions were determined as an
indication of activity after incubating the plate at 37ºC
for 24 hrs for bacteria and at 25ºC for 72 hrs for fungi.
Acetone was included in each plate as negative control
while chloromphenical and fluconazole were used as pos-
itive control for bacteria and fungi respectively. Activity
index for each extract was calculated.
4. Frank Ngonda, et al.: Extraction, characterization and pharmacological evaluation of leaves and root bark of Dalbergiella nyasae (Baker f.)
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Determination of minimum inhibitory concentration
(MIC) and minimum bactericidal/fungicidal
(MBC/MFC)
Minimum inhibitory concentration (MIC) was deter-
mined for each plant extract showing antimicrobial
activity. Modified micro broth dilution method[11]
was fol-
lowed for determination of MIC values. A loopful of the
bacterial and fungal cultures from the slant were inocu-
lated in the nutrient broth and incubated at 37 ± 1ºC for
24 hours for bacteria and at 25ºC for 72 hrs for fungi.
The fresh broth (20 ml) was seeded with 0.25 ml of the
24 hour broth cultures and a two-fold serial dilution
method followed. The extracts were dissolved in acetone
to obtain a 10 mg/ml solution. A 0.2 ml solution of the
plant extract was added to 1.8 ml of the seeded broth to
forms the first dilution. 1 ml of first dilution was diluted
further with 1 ml of the seeded broth to produce the
second dilution, and the process repeated until six such
dilutions were obtained. A set of tubes containing only
seeded broth were kept as control and suitable solvent
controls also maintained. After incubation for 24 hours at
37 ± 1ºC, the tube with no visible growth of the micro-
organism was taken to represent the Minimum Inhibi-
tory Concentration (MIC) of the test sample which was
expressed in mg/ml.
Determination of total activity (TA)
Total Activity is the volume at which test extract can be
diluted with the ability to kill microorganisms. It is cal-
culated by dividing the amount of extract from 1 g plant
material by the MIC of the same extract or compound
isolated and expressed in ml/g.[12]
Phytochemical screening
The plant extracts were screened for the presence of
flavonoids, alkaloids, saponins, terpenoids, steroids
and sterols, tannins and phenols using Sofowora[13]
and
Harborne methods.[14]
Flavonoids were identified by
adding 5 ml of dilute ammonia solution to an aqueous
filtrate of the extract, followed by the addition of con-
centrated sulphuric acid. A yellow coloration observed
in the extract indicated the presence of flavonoids. Alka-
loids were tested by adding a few drops of acetic acid to
5 ml of an extract followed by modified Dragendroff’s
reagent (potassium iodide mixed with bismuth oxyni-
trate) and mixed well. An orange red precipitate formed
indicated the presence of alkaloids. Saponins were tested
by boiling 2 grams of the powdered sample in 20 ml of
distilled water in a water bath and filtered. 10 ml of the
filtrate mixed with 5 ml of distilled water and was shaken
vigorously for a suitable persistent froth. The frothing
mixed with 3 drops of olive oil and shaken vigorously,
and formation of emulsion observed indicated the pres-
ence of saponins. Terpenoids detection was carried out
by mixing 5 ml of plant extract with 2 ml of chloroform
and concentrated sulphuric acid added to form a layer. A
reddish brown coloration of the interface formed showed
the presence of terpenoids. Steroids and sterols were
identified by adding 2 ml of acetic anhydride to 0.5 g of
plant extract with 2 ml of sulphuric acid. Colour change
from violet to blue green in the sample indicated the pres-
ence of steroids and sterols. Tannins were identified by
boiling 0.5 gram of the powdered sample in 20 ml of
distilled water in a test tube and then filtered. A few drops
of 0.1% ferric chloride added and observed for brownish
green or a blue-black coloration indicated the presence of
tannins. And phenols were identified by adding few drops
of drops of methanol and ferric chloride solution to the
plant extract mixed. A blue green or red colour indicated
the presence of phenol.
Determination of phytoconstituents by high
performance liquid chromatography (HPLC)
Further determination of phytoconstituents were per-
formedusingaShimadzoLC-10ADHPLCsystem(Japan),
equipped with a Shimadzo SPD-10AV UV-VIS Spectro-
photometer detector with a thermostted flow cell and a
selectable two wavelengths of 190–370 nm or 371–600 nm
with SCL-10A system control. The detector signal was
operated at ultraviolet wavelength detection at 254 nm.
An Agilent ZORBEX SB-C18 (Agilent Technologies,
USA) column (3.5 µm, 4.6 mm × 150 mm, i.d.) was used
for chromatographic separation. The injection volume of
20 µL was used. The isocratic mobile phase comprised
methanol: acetonitrile (60:40). Analysis was performed at
a flow rate of 0.6 mL/min. 10 mg of the plant extracts
were dissolved in appropriate solvents filtered through
Whatmann paper No. 1 into volumetric flask and made
up to 25 ml.
RESULTS
Antimicrobial activity (assessed in terms of inhibition
zone and activity index) of the plant extracts tested against
different microorganisms were recorded in Table I. In the
present study, 6 crude extracts of acetone, ethanol and
waterand8differentfractionsof dichloromethane,hexane,
n-butanol and ethyl acetate were tested for their bioactiv-
ity. 4 crude extracts and 2 fractions showed some signifi-
cant inhibitions against the test microorganisms. In this
investigation, the leaves extracts showed strong bioactivity
5. Frank Ngonda, et al.: Extraction, characterization and pharmacological evaluation of leaves and root bark of Dalbergiella nyasae (Baker f.)
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compared to the root bark extracts for both crude and
fractions and the most susceptible microorganisms were
P. aeruginosa and S. aureus. Excellent antibacterial activity
against S. aureus were observed in n-butanol fraction of
leavesextract(IZ21mm,1.16±0.05,MIC0.31mg/ml)and
ethanol extract (IZ 20 mm, 0.77 ± 0.05, MIC 1.25 mg/ml).
ForE.coli,bestantibacterialactivitywasrecordedinn-buta-
nol fraction of leaves extract (IZ 18 mm, 0.67 ± 0.17, MIC
0.31 mg/ml). The excellent antifungal activity against C.
albicans was also observed in n-butanol fraction of leave
extract (IZ 21 mm, 0.70 ± 0.14, MIC 0.16 mg/ml). In the
root bark, best antibacterial activity against S. aureus were
observed in ethanol extract (IZ 15 mm, 0.58 ± 0.13, MIC
0.31 mg/ml) and against P. aeruginosa were observed in ace-
tone extract (IZ 15 mm, 0.48 ± 0.06, MIC 0.31 mg/ml).
For E. coli, best antibacterial activity was recorded in etha-
nol extract (IZ 12 mm, 0.54 ± 0.05, MIC 0.625 mg/ml).
The water extract and hexane fractions possessed the least
bioactivity against the test microorganisms.
The range of MIC and MBC/MFC of extract (Table II)
evaluated for bioactivity in diffusion assay recorded were
0.31–2.5 mg/ml and 0.625–5 mg/ml, 0.31–5 mg/ml and
0.625–10 mg/ml, 0.16–5 mg/ml and 0.625–10 mg/ml,
0.16–1.25 mg/ml and 0.625–2.5 mg/ml for E. coli, S. aureus,
P. aeruginosa and C. albicans respectively. In the present inves-
tigation, lowest MIC value 0.16 mg/ml were recorded
against Gram negative P. aeruginosa and one yeast species
C. albicans respectively whereas, against Gram positive
S.aureusandE.coliMICvalue0.31mg/mlwasalsorecorded,
showing significant antimicrobial potential of test extracts.
Amount of extracts isolated from plant parts and total
activity (TA) were calculated and recorded in Table IIIa
and IIIb. Total activity is the volume at which test extract
can be diluted with the ability to kill microorganisms.
The maximum TA value obtained from acetone, distilled
water and ethanol crude extracts were 95.4, 126.72 and
293.12 ml/g against E. coli, P. aeruginosa and C. albicans
respectively. The best solvent in extracting compounds
was ethanol (183.3 mg/g) and distilled water (39.6 mg/g)
in leaves and root bark respectively. Most of the extracts
recorded slightly high TA values against the test organ-
isms even at low concentration indicating the potential to
inhibit the growth of microorganisms.
The acetone extract was resolved into 6 different fractions as
shown in a schematic representation of the solvent-solvent
resolution in Figure 1. The ethyl acetate and hexane had the
greatest quantity of the material from the crude acetone
extract with a percentage yield of 49% and 24% while water
and butanol had least materials with a yield of 1% and 11%
for leaves and root bark extracts respectively. The recovery
of the fractions from the origin crude extract was 67% and
40% for leaves and root bark extracts respectively.
The qualitative phytochemical screening of the leaves and
root bark extracts showed the presence of alkaloids, fla-
vonoids, saponins, terpenoids, steroids and phytosterols,
tannins and phenolic compounds as in Table IV. Alka-
loids, flavonoids, saponins were more abundant in leave
extract compared to root bark extract; this confirms the
presence of the phytoconstituents responsible for antimi-
crobial activity in the leaf and root bark extracts.
The best way for chemical characterization is HPLC fin-
gerprint analysis as it provides qualitative and quantitative
information on the herbal plant extracts.[15]
In the pres-
ent investigation, analysis of ethanol extracts of leaves
and root bark showed 10 fingerprint profiles peaks of
phytoconstituents as indicated in Figure 2 and 3. The fin-
gerprint profile of leaves extract recorded major peak at
1.68 min (89.76%) and minor peaks at 7.35 min (3.94%),
6.43 min (1.71%) and 3.34 min (1.21%) whereas root bark
extract recorded 2 major peaks at 1.76 min (34.06% and
1.96 min (61.50%) and minor peak at 3.32 min (1.66%). In
this investigation, the profiles showed that proportion of
phytoconstituents present were more abundant in leaves
(20,773,550) as compared to the root bark (3,063,700)
confirming the earlier findings as in Table 3a.
DISCUSSION
Results of the present study showed that ethanol
and acetone extracts from leaves and root bark and
Figure 3. HPLC fingerprint rootbark.
6. Frank Ngonda, et al.: Extraction, characterization and pharmacological evaluation of leaves and root bark of Dalbergiella nyasae (Baker f.)
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dichloromethane, ethyl acetate and n-butanol fractions
of leaves extracts tested inhibited the growth of selected
bacteria and fungi, indicating the broad spectrum bio-
active nature of the D. nyasae herbal plant. E. coli and
P. aeruginosa are two organisms that causes infections that
are very difficult to combat[16]
and were found to be more
susceptible to the acetone and ethanol extracts and dichlo-
romethane and n-butanol fractions of the leaves extracts.
Excellent antimicrobial activity was demonstrated by
n-butanol fraction of leaves extract by showing low MIC
and MBC/MFC values for P. aeruginosa and C. albicans.
In this investigation, MBC/MFC values were found
higher than the MIC values of the extract against E. coli,
S. aureus, P. aeruginosa and C. albicans respectively indicating
that the extracts had a bacteriostatic/fungistatic effect on
the tested microorganisms. One yeast species C. albicans
was the most susceptible organism in the investigation
after Gram positive bacteria S. aureus and Gram negative
E. coli and P. aeruginosa. Susceptibility difference between
Gram positive and Gram negative may be due to cell wall
structural differences between them. The Gram negative
bacterial cell wall outer membrane appears to act as a bar-
rier to many substances.[17]
The qualitative phytochemical screening of the leaves
and root bark extracts showed different phytoconstitu-
ents composition of alkaloids, flavonoids, saponins,
terpenoids, steroids, quinines, tannins and phenolic com-
pounds. Flavonoids are known to be synthesized by the
plants in response to microbial infection in nature and in
this investigation they were found to be present in both
the leaves and root bark of D. nyasae therefore it is not
surprising that the plant extracts are very effective against
a wide array of microorganisms.[18]
Their activity is prob-
ably due to their ability to complex with extracellular and
soluble proteins and other components of cell walls.
Saponins were reported to be present and have detergent-
like properties and their mechanism of action against
microbes would appear to involve the formation of com-
plex with sterols in the plasma membrane, thus destroying
the cellular semi-permeability and leading to death of the
cell/microbes.[19]
An important aspect of plant extract and
their components is their hydrophobicity which enables
them to partition the lipids disturbing the cell structures
and rendering them more permeable. And extensive leak-
age from microbial cell or the exit of critical molecules
and ions will lead to death of microbial.[20]
In the present study, most of the extracts recorded MIC
values which were significantly lower indicating strong
bio efficacy. It can be noted from the investigation that
the phytochemical screening and antimicrobial activi-
ties observed with the acetone and ethanol extracts and
dichloromethane ethyl acetate and butanol fractions sug-
gest the presence of bioactive compounds which can
serve as antimicrobial agent or lead compounds of an
effective and less toxic antimicrobial agents.
CONCLUSION
The Dalbergiella nyasae leaves and root bark extracts and
fractions displayed potent and relevant pharmacological
activities with considerable antibacterial and antifungal
activity against selected Gram positive and Gram nega-
tive bacteria and fungi. The results obtained show that
the compound could be of pharmaceutical interest for
therapeutical application as complementary antibacte-
rial and antifungal agents in infectious disease. The fin-
gerprint profile obtained by HPLC can further be used
for identification of phytoconstituents understudy. This
research study provide new scientific knowledge about
the Dalbergiella nyasae based on its antimicrobial potential
and phytochemical profiling which has not been reported.
Therefore, further work is recommended on crude
extracts and fractionation of bioactive compounds to
unravel its structures and indicate their exact potential to
inhibit several pathogenic microbes and development of
a novel broad spectrum antimicrobial herbal formulation.
Also, further evaluation of the cytotoxicity effect of the
compounds and extracts needs to be undertaken to justify
claimed use by traditional healers to treat various diseases.
ACKNOWLEDGEMENT
Grateful thanks goes to the Departments of Chemistry
and Biological Sciences, University of Malawi for provid-
ing the facilities for research work. The authors also thank
Profs John Saka, Jane Morris and the entire SABINA
project team for providing the invaluable help during the
research work.
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Phcog J | Dec 2012 | Vol 4 | Issue 34 1
O R I G I N A L A R T I C L EP H C O G J
ABSTRACT
Introduction: Dalbergiella nyasae (Baker f.) belongs to the family Papilionaceae, small to medium sized herbal
plant well known for its medicinal properties in the treatment of gastrointestinal infections including diarrhoea.
This study aimed to determine the in vitro antimicrobial activity of crude extracts and fractions from leaves and
root bark of D. nyasae against selected bacteria and yeast of gastrointestinal relevance. Methods: Fourteen
extracts from leaves and root bark of D. nyasae have been screened for the antimicrobial activity against three
bacteria species including Gram negative E. coli and P. aeruginosa, Gram positive S. aureus and one yeast species
C. albicans using agar well diffusion, micro broth dilution and Total Activity (TA) analysis methods. Crude extracts
have been qualitatively screened for the presence of phytoconstituents and HPLC fingerprint profiles determined.
Results: Excellent antibacterial and antifungal activity against Gram positive S. aureus and one yeast species
C. albicans respectively were observed in n-butanol fraction of leaves extract. In root bark, best antibacterial activity
against S. aureus was observed in ethanol extract while against P. aeruginosa was observed in acetone extract.
For E. coli, best antibacterial activity was recorded in ethanol extract. Phytochemical analysis demonstrated the
presence of alkaloids, flavonoids, saponins and terpenoids. The HPLC fingerprint profiles of leaves extract recorded
one major peak whereas root bark extract recorded two major peaks. Conclusion: This investigation established
a good support for use of D. nyasae plant by traditional healers in Malawi as herbal medicine for gastrointestinal
infections and a base for development of novel potent drugs and phytomedicine.
Keywords: Agar well diffusion, In vitro antimicrobial activity, micro broth dilution, phytomedicine, Total Activity
(TA) analysis.
Extraction, characterization and pharmacological
evaluation of leaves and root bark of Dalbergiella
nyasae (Baker f.)
Frank Ngonda,*¹ Zakalia Magombo,³ Placid Mpeketula¹ and Jonas Mwatseteza²
¹Department of Biological Sciences, Chancellor College, University of Malawi, P.O. Box 280, Zomba, Malawi
²Department of Chemistry, Chancellor College, University of Malawi, P.O. Box 280, Zomba, Malawi
³National Herbarium and Botanic Gardens of Malawi, P.O. Box 528, Zomba, Malawi
Submission Date: 27-9-2012; Review completed: 10-11-2012; Accepted Date: 4-12-2012
INTRODUCTION
Globally, over 10 million under five children die every
year mostly from bacterial and fungal related infections
(diarrhoea, pneumonia, meningitis, etc) and about 90%
of these children come from the developing countries.[1]
And the major cause of this morbidity and mortality in
the developing countries includes among others poor
availability of interventions and spread and emergency
of antimicrobial resistance.
Multiple drug resistance has become a real problem in
pharmacotherapeutics due to an increasing number of
diseases exhibiting various levels of drug resistance.[2]
Furthermore, the development of synthetic drugs has
slowed down as a result of drug resistance.[3]
Conse-
quently, this has created a new renewed interest in the
search for new drugs in order to combat resistance.
*Corresponding author.
Frank Ngonda
Department of Biological Sciences, Chancellor College
University of Malawi, P.O. Box 280, Zomba, Malawi
E-mail: ngondafb@yahoo.com
Placid357@yahoo.com
jfmwatseteza@cc.ac.mw
zlkmagombo@hotmail.com
DOI:](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)