HIT 200 PROJECT

Group members

Lionel M Ferenando H180421C

Talent Mhlanga H180017V

Tinotenda B Chiwanza H180404G

Gamuchirai L Bete H180725J

Tinotenda G Zvinoira H180021G

Fiona Mutsambiwa H180726M

Tanyaradzwa Nyakurerwa H180161B

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EVALUATION OF
ANTIBACTERIAL AND
ANTIFUNGAL OF
DALBERGIELLA nyasae

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Title
EVALUATION OF ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF
Dalbergiella nyasae (MUSWATI) BARK

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Tinotenda Chiwanza, Tanyaradzwa Nyakurerwa, Gamuchirai Bete, Fiona Mutsambiwa, Talent

Mhlanga, Tinotenda Grace Zvinoira and Lionel Ferenando

Bachelor of Pharmacy [Part 2]

Pharmaceutical Technology Department

Harare Institute of Technology

15015 Ganges Road, Belvedere

Harare, Zimbabwe

2020

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Published by the Group five 2020 HIT 200

For Harare Institute of Technology
P.O. Box BE277 Belvedere, Harare Zimbabwe
Pharmaceutical Technology Department Harare Institute of Technology 15015 Ganges Road,
Belvedere Harare, Zimbabwe

© Harare Institute of Technology 2020

This is a trade mark of HIT.
HIT is the university which is spearheading innovation through application of knowledge and
entrepreneurship through team building projects like this one.
First published 2020
Design and layout by Ferenando Graphical Designs
Printed in Zimbabwe by Tsheda Printing Company, Harare
ISBN 978 0 85369 727 5
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system,
or transmitted in any form or by any means, without the prior written permission of the
copyright holder.
The publisher makes no representation, express or implied, with regard to the accuracy of the
information contained in this book and cannot accept any legal responsibility or liability for any
errors or omissions that may be made.
The right of Harare Institute of Technology to be identified as the author of this work has been
asserted by him in accordance with the Copyright, Designs and
Patents Act, 1988.

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Declaration
The author and publisher have done everything possible to make this book accurate, up to date,
and in accord with accepted standards at the time of publication. The author, editors, and
publisher are not responsible for errors or omissions or for consequences from application of the
book, and make no warranty, expressed or implied, in regard to the contents of the book. Any
practice described in this book should be applied by the reader in accordance with professional
standards of care used in regard to the unique circumstances that may apply in each situation.
This book contains information obtained from authentic and highly regarded sources.
Reasonable efforts have been made to publish reliable data and information, but the author and
the publisher cannot assume responsibility for the validity of all materials or for the
consequences of their use.
Information in this publication have been reviewed and evaluated by the panel of experts from
Harare Institute of Technology Pharmaceutical Technology Department represented below.

Professor B. M. Gundidza………………………………………………

Mr. T. Musenda ………………………………………………………

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Dedication

We would like to dedicate this publication to our friends, families and the community at large
who worked so hard for us to be where we are today. Their support in our lives for us to achieve
whatever we have today has made this publication possible. We are all saying thank you and we
love you.

Tinotenda Chiwanza, Tanyaradzwa Nyakurerwa, Gamuchirai Bete, Fiona Mutsambiwa, Talent

Mhlanga, Tinotenda Grace Zvinoira and Lionel Ferenando

July 2020

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Acknowledgements

A publication containing so much detail could not be produced without the help of a number of
pharmaceutical scientists at Harare Institute of Technology.
Grateful thanks go to Professor B. M. Gundidza of Harare Institute of technology who helped us
with information of some medicinal plants we used and we can best extract and prepare them.
Mr. T. Musenda of Harare Institute of Technology helped us with the idea development and the
formulation of the final products. The guidance provided on the research and documentation is
greatly appreciated.
Thanks are also extended to Mr. Chaweza at Harare Institute of Technology Department of
Pharmaceutical Technology for his support and assistance in all the laboratory work that was
undertaken.
Thanks are also gratefully extended to the staff of the Pharmaceutical Technology Department
of Harare Institute of Technology who assisted for this publication to come out, we hope, to
express their thoughts clearly, concisely, and accurately.

Tinotenda Chiwanza, Tanyaradzwa Nyakurerwa, Gamuchirai Bete, Fiona Mutsambiwa, Talent

Mhlanga, Tinotenda Grace Zvinoira and Lionel Ferenando

July 2020

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Table of Contents
.................................................................................................................................................................1
Title.........................................................................................................................................................2
EVALUATION OF ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF Dalbergiella
nyasae (MUSWATI) BARK....................................................................................................................2
Declaration..............................................................................................................................................4
Dedication...............................................................................................................................................5
Acknowledgements.................................................................................................................................6
CHAPTER 1...................................................................................................................................................9
EVALUATION OF ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF Dalbergiella nyasae (MUSWATI)
BARK............................................................................................................................................................9
INTRODUCTION AND BACKGROUND...........................................................................................................9
Medicinal details.........................................................................................................................................9
STUDY OBJECTIVES....................................................................................................................................10
CHAPTER 2.................................................................................................................................................11
LITURATURE REVIEW.................................................................................................................................11
CHAPTER 3.................................................................................................................................................27
Plant description........................................................................................................................................27
Cultivation details......................................................................................................................................28
CHAPTER 4.................................................................................................................................................29
PRACTICAL SCHEDULE...............................................................................................................................29
EXTRACTION OF THE CRUDE DRUG...........................................................................................................29
PRELIMINARY PHYTOCHEMICAL SCREENING.............................................................................................30
PHYSICAL EVALUATION.............................................................................................................................33
Determination of total activity (TA)..................................................................................................35
Determination of ash values..............................................................................................................35
Extractive values...............................................................................................................................36
CHAPTER 5.................................................................................................................................................37
RESULTS, DISCUSSION AND CONCLUSION.................................................................................................37
Antimicrobial activity of Dalbergiella nyasae............................................................................................37
DISCUSSION...............................................................................................................................................39
CONCLUSION.............................................................................................................................................40

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APPENDIX..................................................................................................................................................41
Avondale, Harare, Zimbabwe................................................................................................................41

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CHAPTER 1
EVALUATION OF ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF
Dalbergiella nyasae (MUSWATI) BARK

INTRODUCTION AND BACKGROUND

Globally, the attractions that have led to the study of medicinal plants as a basis of
pharmacologically active compounds have improved and have become greater than ever before
(Mbolekwa et al’, 2013). Drugs derived from natural sources play a significant role in the
prevention and treatment of human diseases. According to the World Health Organization
(WHO) (2002-2005) medicinal plants are considered the best source to obtain a variety of drugs
which can be used as herbal medicines, safety should also be checked before use. About 80% of
individuals from developed countries use traditional medicines, which have compounds derived
from medicinal plants and they are acknowledged as the main sources of medicines to treat
infectious diseases (Ellof, 1998).

The phytochemicals from the crude extracts of known antimicrobial properties are considered to
have great significance in development of therapeutic medicines. Plants produce a wide variety
of secondary metabolites which are used as precursors or as lead compounds in the
pharmaceutical industry (Selvamohan, 2012). Plants have been used because they have
antimicrobial traits which are due to the secondary metabolites synthesized by the plants. The
screening of plants product for anti-microbial activity has shown that the higher plants represent
a potential source of novel antibiotic prototypes (Afolayan, 2003).

The human environment in most African countries is crowded while sanitation is poor and this
provides a fertile ground for disease outbreaks. Diseases like diarrhoea and dysentery which are
caused by bacterial entero-pathogens are among the core causes of morbidity and death (Alanis
et al’, 2005). Plants still make an input that is very important to health care, even though there is
great progress in modern medicine. The reason for this is the growing appreciation of the value
of traditional medical systems, and the recognition of medicinal plants from the native person.

Medicinal details

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Both leaves and the root bark are used in the treatment of a range of gastrointestinal infections
including diarrhea. The plant has been shown to have antifungal and antibacterial activity. Long
before mankind discovered the existence of microbes, the idea that certain plants had healing
potential, indeed, that they contained what we would currently characterize as antimicrobial
principles, was well accepted. Since antiquity, man has used plants to treat common infectious
diseases and some of these traditional medicines are still included as part of the habitual
treatment of various maladies, it has generally been the essential oils of these plants rather than
their extracts that have had the greatest use in the treatment of infectious pathologies in the
respiratory system, urinary tract, gastrointestinal and biliary systems, as well as on the skin,
(Strugnell, 2006).

STUDY OBJECTIVES
 To evaluate antifungal properties of Dalbergiella nyasae (Muswati) bark

 To evaluate antibacterial properties of Dalbergiella nyasae (Muswati) bark

 To compare antifungal and antibacterial effects in dried Dalbergiella nyasae (Muswati)

bark samples in different solvents

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CHAPTER 2
LITURATURE REVIEW

EXTRACTION CHARACTERIZATION AND PHARMACOLOGICAL EVALUATION

OF LEAVES AND ROOT BARK OF Dalbergiella nyasae (Baker f.)
AUTHOR: Ngoda. F, Magombo. Z, Mpeketula. P, Mwatseteza. J, (2012)
A study done by Ngoda. F, Magombo.Z et al' 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. In the study, fourteen extracts from leaves and
root bark of D. nyasae were 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 were qualitatively screened for the presence of
phytoconstituents and HPLC fingerprint profiles determined.
The results showed 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

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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.
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.

ANTI - BACTERIAL GUIDED FRACTIONATION OF DALBERGIA SISSOO

AUTHOR: Subedi G, Sah C.K, Pokharel K.J, Chaudhary M, Aryal P, Bhandari I, and
Bhandari (2017)

In the study of the plant Dalbergia. Sissoo, various chemical tests were done which involved the
evaluation of anti-bacterial activity of D. sissoo leaves and bark extracts of chloroform, ethyl
acetate, acetone and methanol which was done by guided fractionation. The method used was
done using bioassay guided fractionation by disc diffusion on Staphylococcus aureus (gram-
positive) and Escherichia coli (gram-negative) bacteria. Ampicillin for S. aureus and
Norfloxacin for E. coli were used as positive control and DMSO (Dimethyl Sulphoxide) was
used as a negative control. The anti-bacterial activity of methanol extracts of the leaves, were
found to be more as compared to other three extracts. The S. aureus was more susceptible to
plant extracts than E. coli and the anti-bacterial activity against S. aureus was more as compared
to E. coli in both the leaves and barks extracts.

PHYTOCHEMICAL SCREENING, ANTIOXIDANT AND ANTIMICROBIAL

ACTIVITIES OF Dalbergia melanoxylon TREE

AUTHOR: Najeeb T. M, Issa T.O, Mahomed R. H, Ahmed, (2018)

This study was done to investigate and evaluate antimicrobial, phytochemicals, cytotoxicity and
antioxidant activities of leaves and bark of different extracts of Dalbergia melanoxylon.
Standard methods for phytochemical screening and physiochemical properties of the various
extracts of Dalbergia melanoxylon leaves and bark were done. TLC technique was used for

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separation of extracts from the bark, using three solvents methanol, ethyl acetate and water. The
Scavenging effect of DPPH radical values of different extracts was used to evaluate the
antioxidant activities of the extracts. The results obtained showed that Dalbergia melanoxylon
extracts from leaves and bark have flavonoids, tannins, alkaloids, reducing sugar and
glycosides. The highest anti-microbial activity was found in the ethanol extract of the bark
against Bacillus subtilis. Cytotoxicity studies revealed that the ethyl acetate extract of leaves
showed a high mortality activity against Culex larvae while the ethanol extract of the leaves had
the highest antioxidant activity

EVALUATION of ANTIMICROBIAL ACTIVITY and QUANTITATIVE

PHYTOCHEMICAL SCREENING of SOLVENT EXTRACTS of Dalbergia melanoxylon
(Guill. & Perr.)

AUTHOR: Amri E, Juma S, (2016)

Antimicrobial activity of crude petroleum ether, chloroform, methanol and aqueous extracts of
the stem bark, root and leaf of Dalbergia melanoxylon were investigated by Guill and Perr.
Solvent extractions were done through serial extraction with increasing order of polarity of
solvents. Different concentrations ranging from 50 mg/ml to 250 mg/ml of crude extracts of
plant plants were used to antimicrobial activity of Dalbergia melanoxylon. Microbes used for
antimicrobial tests were Staphylococcus aureus, Escherichia coli and Candida albican.
Clotrimazole and Gentamycin were used as positive controls for fungi and bacteria respectively
and Dimethyl sulphoxide (DMSO) was used as a negative control.
The results showed that at high concentration of crude extracts the antimicrobial activity was
high as compared to low concentration. Stem extracts had the highest zone of inhibition
concentration for Staphylococcus aureus followed by leaf extract for Candida albicans which
had the lowest zone of inhibition. Methanol and aqueous extracts had better scores for
antimicrobial activity compared to other solvents. Phytochemical screening revealed the
presence of different phytochemical constituents in different plant parts thus unveiling the
antimicrobial potential of Dalbergia melanoxylon. The ability of the crude extracts of
Dalbergia melanoxylon to inhibit the growth of bacteria and fungi is an indication of its
potential for broad application as antimicrobial agents.

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ANTIOXIDANT, ANTIMICROBIAL PROPERTIES AND PHENOLICS OF

DIFFERENT SOLVENT EXTRACTS FROM BARK, LEAVES AND SEEDS OF
Pongamia pinnata (L.) Pierre

AUTHOR: Zahid I. Sajid, Farooq. A, Ghulam. R, Khalid M. A and Anwarul-Hassan. G,

(2012)

The study conducted by Sajid Z. I, Anwar. F et al’ appraises the antioxidant and antimicrobial
attributes of various solvent extracts (absolute methanol, aqueous methanol, absolute ethanol,
aqueous ethanol, absolute acetone, aqueous acetone, and deionized water) from bark, leaves and
seeds of Pongamia pinnata  (L.) Pierre. The different parts of Pongamia pinnata such as bark,
leaves, seeds, roots, flowers and stem have been used traditionally in the native medicine
systems of different civilizations. The flowers of the Pongamia pinnata were found to have
anti-hyperglycaemic and anti-lipid peroxidation properties. Its bark is used in piles while the
leaves are used as a medicated bath and in rheumatic pains and the seeds in hypertension,
bronchitis, whooping cough, skin diseases and rheumatic arthritis. Also, the seed oil was
extracted alcoholic constituents that showed activity in both Gram positive and Gram-negative
bacteria.

Extraction of the P. pinnata using aqueous methanol (20:80) produced antioxidant components
of 16.31% bark, 11.42% leaves and 21.51% seeds. The barks extract possessed greater levels of
total phenolic than from any other plant extracts having an inhibition of linoleic acid
peroxidation of 69.23% and a higher DPPPH radical scavenging activity. Also, bark extract
tested against a set of bacterial and fungal strains showed the strongest antimicrobial activity
with the largest inhibition zone and lowest minimum inhibitory concentration (MIC). The
HPLC analysis that was conducted of aqueous methanol extracts from bark, leaves and seeds
indicated the presence of protocatechuic, ellagic, ferulic, gallic, gentisic, 4-hydroxybenzoic and
4-hydroxycinnamic acids in bark (1.50–6.70 mg/100 g DW); sorbic, ferulic, gallic, salicylic
and p-coumaric acids in leaves (1.18–4.71 mg/100 g DW); vanillic, gallic and tannic acids in
seeds (0.52–0.65 mg/100 g DW) as the main phenolic acids.

The investigation concluded that the tested parts of P. pinnata, in particular the bark, had strong
potential for the isolation of antioxidant and antimicrobial agents. The study done was the first

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attempt to show the variations of biological activities among bark, leaves and seeds of P.
pinnata using a range of extraction solvents. Aqueous methanol was established to be the most
effective solvent to recover higher amounts of phenolics from different parts of P.
pinnata compared with other solvents. It was also concluded that extracts from bark of this plant
had higher antioxidant and antimicrobial activities and concentration of phenolic acids among
others, regardless of the extraction solvent used. Further detailed studies on the isolation and
therapeutic properties of bioactives of this plant using some in vivo models is recommended to
establish specific applications and to formulate new and potent antimicrobial drugs of natural
origin.

POTENT BACTERICIDAL ACTION OF A FLAVONOID FRACTION ISOLATED

FROM THE STEM BARK OF Butea frondosa

AUTHOR: Mishra. U. S, Chakraborty. P, Dasgupta. A, Dastidar S. G, Martins. M and L.

Amaral, (2009)

The study done describes the antimicrobial property of a flavonoid fraction obtained from the
stem bark of the Butea frondosa plant. The flavonoid fraction isolated from the ethyl acetate
fraction (BF-1) of Butea frondosa (L.) stem bark showed antimicrobial activity when tested
against 129 bacterial strains belonging to 9 different genera of both gram-positive and gram-
negative types. Minimum inhibitory concentration (MIC) of the fraction BF-1 was determined
following NCCLS guidelines using the agar dilution method. This was done by determining the
in vitro inhibitory activity of BF-1 against a large number of species and strains. Twenty-four
out of 36 strains of Staphylococcus aureus and Bacillus spp (33%) were inhibited by 50-200
mg/l of the fraction. At a concentration of 100mg/l activity against 100% of the bacteria,
Shigella spp., Salmonella spp. and even a few Pseudomonas at concentrations between 50-200
mg/l.

Other bacteria including Escherichia coli, Vibrio cholera (20%) at a concentration of 50 mg/l
and V. parahaemolyticus were moderately sensitive to BF-1. The activity of BF-1 against
strains of S. enteritidis NCTC 74 varied (MIC for the bulk of the strains is 200 to 400 mg/l).
The activity of BF-1 against Klebsiella spp. and Proteus spp. took place with a minimum
concentration of 400 mg/l and hence these species were relatively resistant to this fraction. In

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the in vivo studies, this fraction offered significant protection to Swiss albino mice at a
concentration of 80 μg/mouse (p<0.001) when they were challenged with 50 median lethal dose
of Salmonella enteritidis NCTC 74 although the lethality differed with concentration of
different bacteria tested. A fraction named BF-1 that was isolated from an ethyl acetate fraction
of Butea frondosa provided protection against an infection from a Salmonella enteritidis NCTC
strain.

The results of the study showed that a single dose of 80 mg/l of BF-1 protected the mouse from
a lethal infection by S. enteritidis NCTC 74. This low concentration of BF-1 resulted in the
elimination of the Salmonella from the infected animal. Further studies are being done to
completely identify compounds present in the flavonoid fraction as well as to determine any
synergism with antibiotics, antimicrobials and non-antibiotics.

ANTIMICROBIAL ACTIVITY OF THE EXTRACT OF STEM BARK OF Diplotropis

ferruginea Benth

AUTHOR: Cerqueira, G.S, Rocha N, Almeida J, de Freitas A, Lima EO, Filho J, et al,
(2011)

The antibacterial action of the dried powdered stem bark of Diplotropis ferruginea (family:
Fabaceae) was evaluated using several bacteria for example: Staphylococcus aureus,
Escherichia coli and the following fungi: Candida albicans, Candida tropicales
515), Tricophytonrubrum, Tricophytonrubrum, Aspergillusflavus and Fusarium. Standard
antimicrobial agent used as a control was Penicillin G Benzantine.

This is the first report about the antibacterial activity of Diplotropis ferruginea Benth. In this
study, the ethanol extract of D. ferruginea was tested for its antimicrobial activity against strains
gram-positive and gram-negative. In order to determine the minimal inhibitory concentration,
assays were carried out by micro dilution method. The extract was screened for antimicrobial
activity, and it showed antibacterial activity against Escherichia coli and Pseudomonas
aeruginosa. The antibacterial activity of extract and flavonoid isolated from D.
ferruginea presented excellent results against the pathogenic microorganisms tested, and the
extracts presented antibacterial activity against clinically relevant pathogens (gram positive and

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gram negative). D. ferruginea stem bark extract was active against Pseudomonas

aeruginosa and Escherichia coli.

SYNERGIC ANTIFUNGAL AND ANTIBACTERIAL ACTIVITY OF ALCOHOLIC

EXTRACT OF THE SPECIES Robinia pseudoacacia L. (Fabaceae)
AUTHOR: Bita A, Roşu A. F, (2012)

Plant species Robinia pseudoacacia L. (Fabaceae) has been used as a medicinal plant since
ancient times, as its infusions and extracts have antacid, antibacterial, purgative and emenagogic
properties. The volatile oil of the flowers is also used in perfumery and cosmetics. Due to its
high content of volatile oil phenolic compounds, flavonoids and tannins with antimicrobial
properties, the present study proposed to investigate the antibacterial and antifungal effect of the
species Robinia pseudoacacia. The dry powdered flowers, leaves, bark and seeds of Robinia
pseudoacacia were subjected to Soxhlet extraction with 90% ethanol. The alcoholic extract
obtained in the concentration of 100 mg / ml, was tested using sterile discs of Whatman No. 1
filter paper, impregnated with 100 mg extract.

Antibacterial and antifungal effects was evaluated by the Kirby-Bauer disc diffusion method, in
accordance with the NCCLS / CLSI standard, using the following infectious agents isolated
from patients and the corresponding reference strains: methicillin-resistant Staphylococcus
aureus and methicillin-sensitive haemolytic S. epidermidis, Streptococcus pyogenes,
Enterococcus, Enterobacter aerogenes, Escherichia coli, Salmonella choleraesuis, Klebsiella
pneumoniae, Pseudomonas aeruginosa, Proteus, Candida albicans. Extracts from various
different parts of the plant had different antibacterial activities. Extracts of flowers and seeds
were found to be efficient antibacterials for Gram positive cocci. Bark and leaf extracts were
active against Escherichia coli, Pseudomonas, Proteus, Salmonella choleraesuis, Candida
albicans. These results prove the antimicrobial or antifungal properties of certain extracts of
Robinia pseudoacacia L., which offers an alternative treatment with a natural product with
synergistic effects with conventional antibacterial treatment.

ANTIBACTERIAL STUDIES OF THE STEM BARK OF Detarium Microcarpum Guill.

&Perr. (Fabaceae)

AUTHOR: Abubakar S et al. (2017)

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Bacteria isolates comprising Staphylococcus aureus and Escherichia coli were identified to
genera level using the conventional biochemical tests and were further characterized to species
level using the appropriate microscopic procedures and confirmatory tests. The isolates were
then tested for their antibiotic sensitivity against eight different standard antibiotics and against
three samples from the stem bark of Deuterium macrocarpon. The zones of inhibition,
Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and
the Rate of Kill of the three samples were measured. The isolates were generally resistant to
amoxicillin and Augmentin but susceptible to nitrofurantoin, gentamicin and ofloxacin. Most of
the E. coli isolates were resistant to Tetracycline as compared to the samples of S. aureus
isolates. The antimicrobial activity of the extract and fractions of the stem bark of the plant was
compared with that of a standard antibiotic (ciprofloxacin). The traditional medicinal use of the
plant stem bark was justified by the results of this study.

ANTIMICROBIAL ACTIVITY OF Lannea stuhlmanii, Carissa edulis, Combretum

fragrans, Conyza sumatrensis, Ormocarpum trichocarpum, Sida cuneifolia, Plumbago
zeylanica, and Rhoicissus revoilii

AUTHOR: Kouyaté, A.M. and van Damme, P. (2006)

In these plants ethanolic extracts were screened for their antimicrobial activity against Candida
albicans, Escherichia coli, Staphylococcus aureus and Bacillus pumulus. The methanol, ethanol
and aqueous extracts of seven medicinal plants were evaluated for activity against medically
important bacteria such as Staphylococcus sp., Escherichia coli, Klebsiella sp., and
Pseudomonas sp. hanolic. The in vitro antimicrobial activity was performed by agar well
diffusion method and disc diffusion method. The ethanolic and aqueous extracts showed
minimum antimicrobial activity when compared to methanolic extracts. The extract Phyllanthus
niruri showed the maximum activity against Staphylococcus sp. The use of plant extracts with
known antimicrobial properties, can be of great significance in therapeutic treatment.

The human pathogenic bacteria such as Staphylococcus sp., Klebsiella sp., Pseudmonas sp.,
Escherichia coli were maintained in Nutrient agar slant at 4oC. The ethanolic, methanolic and

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aqueous extract were prepared by dissolving 10gm of fine powder of each medicinal plants
separately in 50 ml of ethanol, methanol and water respectively. The contents were kept in a
shaker for 48 h. The extract was filtered and dried in hot air oven at 40oC. Then the extract was
stored under refrigeration at 4oC for further studies. The 20 ml of sterilized Muller Hinton Agar
was poured into sterile petri-plates, after solidification, 100 μl of fresh culture of human
pathogens were swabbed on the respective plates. The discs were kept over the agar plates using
sterile forceps at various concentrations. The antimicrobial activity of ethanol methanol and
aqueous extracts of seven Indian medicinal plants were investigated using disc diffusion method
and agar well diffusion method against human pathogens such as Staphylococcus sp.,
Escherichia coli, Klebsiella sp., and Pseudomonas sp. These four different pathogens have also
tested with commercially available four different antibiotics. All the medicinal plant extracts
used against the pathogenic organisms have showed different degree of antimicrobial activity
against the pathogens.

ANTIBACTERIAL ACTIVITY OF THE STEM BARK EXTRACTS OF Acacia mearnsii

De Wild

AUTHOR: Mbolekwa B et al (2013)

Antibacterial activity different extracts from the stem bark of Acacia mearnsii was measured
against Gram-positive and five Gram-negative bacterial strains which are Staphylococcus
aureus, Staphylococcus epidermidis, Bacillus cereus, Micrococcus kristinae, Streptococcus
faecalis, Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Klebsiella pneumonia and
Serratia marcescens. The extracts assessed include hexane, ethyl acetate, dichloromethane and
methanol. The hexane, ethyl acetate and methanol extracts showed some inhibitory effects on
the selected bacteria. The hexane extract showed some activity against four Gram-positive and
two Gram-negative bacterial strains, but was not active against one Gram-positive and three
Gram-negative bacterial strains. The ethyl acetate extract was effective against all the bacterial
strains used in this study. The methanolic extract was effective against all the Gram-positive
bacterial strains but it was not active against the Gram-negative bacterial strains except
Escherichia coli. The dichloromethane extract was not active at all against all the bacterial
strains tested. Hexane, ethyl acetate, methanol and dichloromethane were obtained from Sigma-

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Aldrich as analytical reagents. The orbital shaker used was an MRC with twelve positions for
250 ml erlen-meyer flasks. The Buchner funnel was a Corning and the filter paper was a
Whatman No. 1. The rotary evaporator was a Buchi R- 215 fitted with a vertical water-cooled
condenser. Nutrient agar was purchased from Arcos.

The antibacterial activity was evaluated using the dilution-in-agar technique (Alanis et al.,
2005). The bacterial species that were used were sub-cultured and preserved on nutrient agar
were contained in Petri dishes. Nutrient agar 60 ml was prepared and sterilized using an
autoclave at 121°C. The hexane extract of the bark of A. mearnsii showed potent antibacterial
activity against E. coli with MIC value of 10.0 mg/ml and the results were almost the same as
those of hexane extracts of the bark of Ficus congensis with MIC values of 8.0 mg/ml. The
extract was not active against K. pneumonia. In the study, the hexane extract showed activity
against four Gram-positive bacterial strains with MIC value of 10.0 mg/ml but was not active
against S. faecalis. This extract also showed some activity against two Gram-negative bacterial
strains, E. coli and P. aeruginosa, but was not active against the Gram-negative bacterial
strains, S. flexneri, K. pneumonia and S. marcescens. The ethyl acetate extract was effective
against all bacterial strains used in this study. MIC values ranged from 1.0 and to 10.0 mg/ml.
The methanolic extract was active against all Gram-positive bacterial strains, it had MIC values
of 1.0 and 5.0 mg/ml. It was not active against the Gram-negative bacterial strains except for E.
coli which was susceptible at MIC value of 5.0 mg/ml. The extract from dichloromethane was
not active at all against any of the bacterial strains tested. It was seen that dichloromethane was
not a suitable solvent for extraction of the active compounds in A. mensi since there were no
active ingredients extracted.

ANTIFUNGAL EFFECTS OF Acacia ataxacantha (Bark)

AUTHOR: Ahmad B, Khan I, Azam S, Bashir S, Ahmad J, Hussain F (2011)

Acacia ataxacantha is a spiny plant in the Fabaceae family. The study aimed to evaluate the
antifungal activities of Acacia ataxacantha against of Aspergillus. Five extracts including
hexane, dichloromethane, ethyl acetate, methanol and mixture of water/ethanol of A.
ataxacantha’s barks were evaluated against Aspergillus strains using agar diffusion method and
counted number fungal spores. Results obtained showed that all the extracts inhibited mycelial

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growth and sporulation of fungi with percentages of inhibition ranging from 5.37- 53.02% and
33.77 to 99.70% respectively. The antifungal activity of the extracts at the same concentration is
marked on the sporulation of fungi. Ethyl acetate and dichloromethane extracts exhibited the
most significant antifungal activity. The overall finding of this study suggests that A.
ataxacantha inhibited the mycelial growth and sporulation of Aspergillus strains. Equally, A.
ataxacantha can be used as an antifungal.
The Potato Dextrose Agar (PDA) plates were prepared by pouring 10 ml of the mixture of
PDA-extract at 1 mg/ml into sterile petri plates. The plates were allowed to solidify for 15 min
and 100 spores prepared in tween (5%) were dropping in the centre of petri dishes which were
then incubated at 25°C. After 5 days, the diameter of mycelia was measured and the number of
spores was counted microscopically using malassez cell. Each test was performed 3 times.
Three petri dishes containing PDA without extract were used as negative control and
Fluconazole (100 μg/ml) was used as positive control. The inhibitory percentage (IP) of extracts
on sporulation ranging from 33.77 to 99.70 % (Table 1). The ethyl acetate extract showed the
strongest activity against A. clavatus with an IP value of 99.70%, while the lowest inhibition
was obtained with the hexane extract (IP = 33.77%) against A. ochraceus. The dichloromethane
and ethyl acetate extracts showed inhibitory effect against all fungal strains tested with an IP
value greater than 70%. The inhibitory effect of the extracts on sporulation was more against A.
parasiticus and A. clavatus (93.14%≤IP ≤99.70%). The IP values of extracts on mycelial
growth range from 5.37 to 47.65% (Table 2). The extracts showed activity against four fungi
including A. parasiticus. The dichloromethane and ethyl acetate extracts showed the highest
activity against A. nidulans with the respective IP values of 53.02% and 51% in comparison to
the reference molecule (64.42%).

ANTIMICROBIAL PROPERTIES OF THE STEM BARK OF Saraca .indica

(Caesalpiniaceae)

AUTHOR: Sainath R, Prathiba J, Malathi R, (2009),

According to SAINATH et al, the antimicrobial activity of stem bark extracts of Saraca.indica
was evaluated against some common bacteria and fungi namely, Staphylococcus aureus,
Escherichia.coli, Pseudomonus.aeruginosa, Bacillus.cereus, Klebsiella.pneumoniae,

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Proteus.mirabilis, Salmonella.typhimurium, Streptococcus.pneumoniae and fungi

Candida.albicans and Cryptococcus.albidus. The antimicrobial activity of the concentrated
extracts was evaluated by using the disc-diffusion method, were the discs were tested in
triplicate, including one with a solvent blank and three for the standard drugs. Methanolic and
aqueous extract exhibited antimicrobial activity with minimum inhibitory concentration (MIC)
ranging from 0.5-2% and 1-3% respectively. Methanolic extract exhibited the strongest activity
against both bacteria and fungi.
ANTIBACTERIAL AND ANTIOXIDANT POTENTIAL OF STEM BARK EXTRACT
OF Bombax.ceiba COLLECTED LOCALLY FROM South Punjab Area of Pakistan

AUTHOR: Masood-ur-Rehman, Naveed Akhtar, and Rehan Mustafa, (2017)

According to Rehman et al, antibacterial and antioxidant activity of methanolic extract of

Bombax.ceibastem bark was evaluated. Phenolic and flavonoid contents were assessed in the
extract. The antioxidant capacity was determined by DPPH, Nitric Oxide scavenging and
reducing power activity. For antibacterial activity, gram negative bacteria (Esherichia.coli,
Pseudomonus.auraginosa and Salmonella.typhi) and gram-positive bacteria (Bacillus.subtilis,
Staphylococcus.aureus) were used. Antibacterial activity of the extract was evaluated by agar
well diffusion method by applying standard inoculums of each strain. The study revealed that
Bombax ceiba displayed inhibitory effect against microbial growth with Salmonella typhi as the
most resistant strain and Staphylococcus.aureusas the most sensitive.

PHYTOCHEMICAL SCREENING ANALGESIC, ANTIMICROBIAL AND

ANTIOXIDANT ACTIVITIES OF BARK EXTRACTS OF Adenanthera pavoninaL.
(Fabaceae).

AUTHOR: Arzumand Ara, Saleh-E-In Md, Nazim A, Meshbahuddin A, Mohamed H. and

Sitesh B. (2010)

In the study of antimicrobial activities of bark extracts of Adenanthera pavonina L. (Fabaceae)

done by Arzumand Ara et al, the disc diffusion method was used to test for antimicrobial
strains. The strains were collected as pure culture and the test samples were made by dissolving
in calculated volumes of solvents separately and applied to sterile discs at different
concentrations and dried to evaporate residual solvents. The test material was placed on nutrient
23
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agar medium with test microorganisms. Kanamycin antibiotic was used as a standard and blank
discs impregnated with solvents were used as positive and negative control respectively. The
experiment was carried out in triplicate and antimicrobial activity was determined by measuring
the diameter of zone of inhibition expressed in millimetre.

The results observed that in the dose of 400ug/disc, the ME showed the strongest inhibitory
activity against (Staphylococcus aureus and Sarcinalutea) which are gram positive and Vibrio
mimicus which is gram negative. The other extract (PE, DCM and ME) showed very weak
inhibitory activity against the entire tested organism at 200ug/disc. The ME extract was
sensitive and all other extracts were insensitive to all tested microorganism. The ME extract
have significant activity indicating the presence of active constituents which are responsible for
this antibacterial activity and may be used for infection treatment (Arzumand Ara et al, 2010)

ANTIMICROBIAL ACTIVITY OF Tamarindus indica Linn

AUTHOR: Doughari J. H, (2006)

The antimicrobial activities of extracts of the stem bark and leaves of Tamarindus indica were
evaluated in the study by JH Doughari since it is used in traditional medicine for the treatment
of cold, fever, stomach disorder, diarrhoea and jaundice and as skin cleanser. The antimicrobial
activity of the aqueous and organic extracts of the plant sample was evaluated by paper disc
diffusion method. The bacterial cultures were adjusted to 0.5 McFarland turbidity standard and
inoculated onto Nutrient agar plates. All the cultures were inoculated into Sabroud Dextrose
agar plates. Bacterial cultures and those of Candida albicans were incubated at 370C for 18h
and other fungal activities at 320C for 48h. Ciprofloxacin and Cotrimoxazole antibiotics were
used and Nystatin and Amphotericin B for fungi were used as standard antimicrobials for
comparison. The Minimum inhibitory concentration (MIC) of the extracts was estimated.

The acetone extracts of stem bark against Proteus mirabilis demonstrated the highest activity
and the water extract against Staphylococcus aureus demonstrated the lowest activity. The
minimum inhibitory concentration and minimum bactericidal concentration results showed that
Staphylococcus aureus had highest MIC (18mg/ml) and MBC (17.5mg/ml), while the lowest

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MIC of 8mg /ml was shown by Salmonella paratyphoid and Bacillus subtilis. The
demonstration of antimicrobial activity by water extracts provides the scientific basis for the use
of these plants as traditional treatments (JH Doughari, 2006)

ANTIBACTERIAL AND ANTICANCER ACTIVITY OF ETHNOMEDICINAL

PLANTS USED IN THE Jongilanga Community, Mpumalanga

AUTHOR: Lall N, Canha M. N, Reid A, Gasa N, Twilley D, Hamilton C and Mahore J

(2018)

The A375 cell line, Propionibacterium acnes (ATCC 11287), Mycobacterium tuberculosis
(H37Rv), in MGIT media and Mycobacterium smegmatis were used in the study of different
plant extracts. The plant material was dried, ground to fine powder, macerated in distilled
ethanol and extracted. The antimicrobial activity was performed according to Eloff (1998).
Propionibacterium.acnes was maintained on sterile nutrient agar plates and sub cultured and
treated to determine the minimum inhibitory concentration. Extracts were dissolved in 10%
DMSO, the 0.5 McFarland Standard and tetracycline were used. The study showed that most
plant family used was Fabaceae and it was not surprising that most of the active species against
P. acnes came from this plant family as they are used in the treatment of a number of skin
disorders including wound healing, infectious diseases, sores or ulcers, skin irritation, burns and
inflammatory skin conditions. Dalbergia melanoxylon and M. sericea showed lowest MICs
against P. acnes. MIC obtained against M. smegmatis was found to be lower than against M.
tuberculosis. The Fabaceae/Leguminosae showed the most promising activity against species of
mycobacterium and the family is most important due to its edibility and medicinal properties.
(Lall N et al. 2018)

ANTIBACTERIAL ACTIVITY OF DIFFERENT SOLVENTS OF Albizia amara

AUTHOR: Shubha, G, Govindaraju, Satyanarayan N and Manjunatha (2014)

The Albizia amara bark, flower, pod and leaf was studied for antibacterial activities in India.
The plant material was washed, sprayed with ethanol and dried under shade and powdered in a
blender. The powdered plant material was subjected for extraction by Soxhlet apparatus using
solvents of different polarity. The in vitro antibacterial activity was carried out against selected
gram positive (Staphylococcus aureus, Bacillus subtilus, Streptococcus haemolytius) and gram
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negative (Salmonella typhi, Escherichia coli, Paeudomonas aerufinosa, Klebsiella pneumonia)

bacterial strains. The assays indicated that the bark of Albizia amara has greater degree of
activity and hence active compound concentration in this extract and this might be due to the
presence of different alkaloids present in the plant. (Shubha G et al 2014)

ANTIMICROBIAL ACTIVITY OF Croton macrostachyus STEM BARK

EXTRACTS AGAINST SEVERAL HUMAN PATHOGENIC BACTERIA

AUTHOR: Obey J.K, von Wright A, Orjala J, Kauhanen J, and Tikkanen-Kaukanen C

(2016)

The leaves and roots from Croton macrostachyus in Kenya are used for traditional medicine in
treating infectious diseases like typhoid and measles, however no antimicrobial activity of the
stem bark is known to exist. In the study, the antibacterial and antifungal effects of methanol,
ethyl acetate and butanol extracts, and purified lupeol of C. macrostachyus stem bark were
determined against common important human gram-negative pathogens like Escherichia coli,
Salmonella typhi, Klebsiella pneumoniae, and Enterobacter aerogenes, the gram-positive
Listeria monocytogenes, and the fungus Candida albicans. The ethyl acetate extract had the
most promising broad scale antimicrobial activity of all the previously mentioned pathogens. It
induced the zone of inhibition between 10.1 ± 0.6 mm and 16.0 ± 1.2 mm against S. typhi, E.
coli, K. pneumoniae, E. aerogenes, and L. monocytogenes with weaker antimicrobial activity
against C. albicans with a zone of inhibition of 5.6 ± 1.0 mm. Amoxicillin, ciprofloxacin,
ampicillin, benzylpenicillin, clotrimazole, and cefotaxime which were the antibiotic controls
that indicated antimicrobial activity with zones of inhibition within the range of 13.4 ± 0.7 to
22.1 ± 0.9 mm. The ethyl acetate extract had a MIC in the range of 125–250mg/mL against all
the bacteria studied and the MIC against C. albicans was 500mg/mL. The obtained results gave
adequate evidence that the use of C. macrostachyus stem bark as a source for antimicrobials
traditionally is effective and that C. macrostachyus stem bark lupeol is a promising
antimicrobial agent against several important human pathogens.

STUDY ON ANTIBACTERIAL ACTIVITY OF THE BARK OF Garcinia lanceifolia

Roxb.

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AUTHOR: Bora, N, S, Kakoti B.B, and Gogoi B (2014)

Garcinia lanceifolia Roxb is an endemic and important medicinal plant that has been used by
various ethnic communities of Northeast India to treat various disorders like dysentery,
dyspepsia, and biliousness. The plant is considered to contain much medicinal value and is
eaten raw or made into pickles by the local people. The present study evaluates the antibacterial
activity of the methanolic extract of the bark of Garcinia lanceifolia which will lead to a
scientific evidence of the use of this plant in cases of dysentery and diarrhoea for its
antimicrobial activity. The methanolic extract of the bark of the plant Garcinia lanceifolia has
shown significant antibacterial activity against four strains of bacterium, namely, Bacillus
subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. Garcinia
lanceifolia bark is seen to have more activity against gram positive bacteria than gram negative
bacteria and in a dose dependent manner. Garcinia lanceifolia bark has important anti-bacterial
activity and may be used to treat various disorders like dysentery, dyspepsia, and biliousness

INVESTIGATION OF ANTIMICROBIAL ACTIVITY OF SOME SELECTED WEED

SPECIES

AUTHOR: Khan W.M, Ali S, Khan M.S, Akhtar N, Ali K and Khan H (2016)

Medicinal plant species are of greater importance as they are dependable source for curing of
various disease conditions. The medicinal value of plant species increases due to different
chemical constituents which are present that bring about different pharmacological actions on
the human body. The methanol leaf extract of Ziziphus jujuba, Withania coagulens, Tinospora
cordifolia, Abutilon indicum and Acacia modesta showed an active antibacterial activity against
Escherichia coli, Pseudomonas fluorescence, Bacillus subtilis, Staphylococcus aureus,
Xanthomonas axonopodis and X. malvacearum. Alternatively, the extract of plants also shows a
significant antifungal activity against Aspergillus flavus, Drechslera turcica and Fusarium
verticillioides as compared to bark and root extracts. The leaf extract of Abutilon indicum and
Acacia modesta showed substantial antibacterial activity against B. subtilis while the leaf
extract which of Z. jujuba showed great results against X. axonopodis and X. malvacearum.
Extract obtained from root and stem of Abutilon indicum showed its antibacterial activity
against all the test bacteria. The leaf and bark extracts of A. modesta showed a progressive result

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against fungal activity of A. flavus. Beside A. Modesta, the Tinospora cordifolia and Z. jujuba
showed active activity against fungi while the methanolic extract of Abutilon indicum showed
an important antifungal activity against F. verticillioides.

CHAPTER 3
Plant description
Dalbergiella nyasae, also known as Mane-pod (English) Munyamhanji, Munyenza, Muswati or
Muvengahonye (Shona), is a small to medium-sized deciduous tree, which belongs to the family
Papilionaceae, and species Fabaceae, (Brummitt, 2007). It has a dense, irregular crown. It is a
profusely-flowering plant, which can grow 4 – 9 metres tall. The leaves of this tree are crowded

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near the ends of branches, and they are imparipinnate with 6-9 pairs of oval leaflets and a
terminal leaflet. The bark is grey-brown in colour, and is longitudinally fissured. Flowers in this
tree are produced in profusion before the leaves in long many-flowered sprays, whitish-cream to
pink with a dark mauve centre. The tree stems and flower stalks are covered in dense brown
velvety hairs. Dalbergiella nyasae has flattened pod, oblong and distinctly fringed with a mane
of long brown hairs, (Burrows, 2005).

Its habitat is not well recorded, but probably in deciduous woodland and bush land; at elevations
from 350 – 1 250 metres. Its flowering time is usually between August- October. Dalbergiella
nyasae is mostly found in Tanzania, Mozambique, Malawi, Zimbabwe and Zambia (Lotter,
2018). In Zimbabwe the tree is found in the Eastern Highlands of the country.

Cultivation details
Dalbergiella nyasae grows best in a sunny position. It succeeds in poor, stony soils. The flowers
are fragrant. This species has a symbiotic relationship with certain soil bacteria; these bacteria
form nodules on the roots and fix atmospheric nitrogen. Some of this nitrogen is utilized by the
growing plant but some can also be used by other plants growing nearby, (Mapaura, 2004).
Propagation of the tree is done with seeds.

Dalbergiella nyasae tree The flowers The bark

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CHAPTER 4
PRACTICAL SCHEDULE

EXTRACTION OF THE CRUDE DRUG

1. Dalbergiella nyasae bark was freshly collected in January 2020 in Masvingo, Bikita.
The plant was authenticated by Professor M. Gundidza and the National Herbarium. The
Botanic Garden confirmed that the plant was Dalbergiella nyasae.

2. After collection of the bark, it was shade dried for a month. It was then cut and ground
into small pieces using a pestle and mortar.

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3. A problem we encountered was that it was difficult to grind the cut pieces of the bark
into fine powder, and some of the powder was blown away by the wind during the
grinding process.

4. After grinding, the biomass from the bark was weighed using a balance and 700g was
placed in one amber bottle, and another 700g in another amber bottle.

5. 1.5L of chloroform and methanol (mixed in the ratio 1:1) was added to one amber bottle,
and 1.5L of acetone was added in another amber bottle. These solvents were used for
extraction of the plant material.

6. According to a study done by Ngoda. F, Magombo et al, 50% of the plant material in the
family Fabaceae was extracted using methanol, proving it to be a good solvent.

7. Other solvents that proved popular include ethyl acetate, di-chloromethane, acetone, and
n-hexane amongst others in several articles. Hence, we used methanol and chloroform
(mixed in the ratio 1:1) and acetone as solvents for our extraction which were available
in our school laboratory.

8. The extraction of the plant material took 7 days, and the filtrate was filtered through a
filter paper. The filtrates were then evaporated under reduced pressure at 87°C using a
rotary evaporator to yield the crude extract and the crude extract was collected in a vial
for further use.

9. Weighing was done again to obtain total yield of crude extract, followed by tests for
secondary metabolites as part of standardization.

PRELIMINARY PHYTOCHEMICAL SCREENING

Aim(s): To determine the phytochemical constituents present in Dalbergiella nyasae

The acetone extract and methanol chloroform extract of Dalbergiella nyasae bark was subjected
to preliminary phytochemical screening of various plant constituents (Harborne, 1998; Kokate,
2001).

Test for Alkaloids

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(i) Dragendroff’s test

Dragendroff’s reagent:

8grams of Bi (NO3)3.5H2O was added in a beaker and dissolved in 20 ml HNO3. 2.72 g of

potassium iodide was added in 50 ml of H2O. The solutions were mixed and allowed to stand
until KNO3 crystals were formed. The supernatant was decanted and made up to 100 ml with
distilled water.

Procedure:

To 0.5 ml of the extract, 2 ml of HCl was added. To this acidic medium, 1 ml of Dragendroff’s
reagent was added. An orange or red precipitate was produced which indicated the presence of
alkaloids.

(ii) Wagner’s test

Wagner’s reagent:

1.2 g of iodide and 2.0 g of potassium iodide were dissolved in 5 ml of sulphuric acid and
diluted to 100 ml.

Procedure:

10 ml of the extract was acidified by adding 1.5% v/v of HCl and a few drops of Wagner’s
reagent. Formation of yellow or brown precipitate confirmed the presence of alkaloids.

(iii) Mayer’s test

Mayer’s reagent:

1.36g mercuric chloride was dissolved in 60 ml of distilled water. 5 g of potassium iodide was
dissolved in 10 ml of water. The two solutions were mixed and diluted to 100 ml with distilled
water.

Procedure:

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1.2 ml of the extract was poured into a test tube. 0.2 ml of dilute hydrochloric acid was added
and 0.1 ml of Mayer’s reagent. A yellowish buff coloured precipitate confirmed the presence of
alkaloid.

Test for Flavonoids

(i) Shinoda’s test:

0.5ml of extract was placed in a test tube. 5-10 drops of diluted HCl was added and small piece
of ZnCl or magnesium. The solution was boiled for a few minutes. In the presence of
flavonoids, reddish pink colour was observed.

(ii) Alkaline Reagent Test:

To 1.0 ml of the extract, a few drops of dilute sodium hydroxide were added. An intense yellow
colour was produced in the plant extract, which became colourless on addition of a few drops of
dilute acid indicating the presence of flavonoids.

Molisch’s test:

The Filtrate was treated with 2–3 drops of 1% alcoholic α-naphthol solution. 2 ml of Conc.
H2SO4 was added along the sides of the test tube. Appearance of brown ring at the junction of
two liquids showed the presence of carbohydrates.

Test for Glycosides

The extract was hydrolysed with HCl for few hours on a water bath and the hydrolysate was
subjected to Legal’s or Borntrager’s test to detect the presence of glycosides.

(i) Legal’s test:

To the hydrolysate, 1 ml of pyridine was added, and a few drops of sodium nitroprusside
solutions were added to make it alkaline with sodium hydroxide solution. Appearance of pink to
red colour showed the presence of glycosides.

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(ii) Borntrager’s test:

The hydrolysate was treated with chloroform, then the chloroform layer was separated. Equal
quantities of dilute ammonia solution were added. Ammonia layer acquired pink colour,
showing the presence of glycosides.

Test for Saponins

The extract was diluted with 20 ml of distilled water and was agitated in a graduated cylinder
for 15 min. The formation of 1 cm layer of foam showed the presence of saponins. 1 ml of the
extract was treated with 1% lead acetate solution. Formation of white precipitate indicated the
presence of saponins.

Test for Tannins

(i) Ferric chloride test:

A few drops of 5% aqueous FeCl3 solution were added to 1-2 ml of the extract. A violet colour
formation indicated the presence of tannins.

(ii) Lead acetate test

A few drops of 1% lead acetate were added to a test tube containing about 5.0 ml of the extract
and. A yellow precipitate indicated the presence of tannins. 5 ml of the extract was treated with
1 ml of 10% aqueous potassium dichromate solution. Formation of yellowish brown precipitate
indicated the presence of tannins.

Test for Proteins and Amino Acids:

1.0 ml of the extract was treated with a few drops of Ninhydrin reagent (Triketo hydrindene
hydrate). Appearance of purple colour showed the presence of amino acids.

(i) Ninhydrin test (ii) Biuret test:

Equal volumes of 5% NaOH solution were added and 1% copper sulphate solution to 1.0 ml of
the extract. Appearance of purple colour showed the presence of proteins.

Test for Anthraquinones

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5 ml of the extract solution were hydrolysed with dil. H2SO4. The solution was extracted with
benzene and 1 ml of dilute ammonia was added. Rose pink colouration indicated the positive
response for anthraquinones.

PHYSICAL EVALUATION

Physical contents such as elasticity in fibres, viscosity of drugs containing gums, selling factor
for mucilage containing materials, froth number of saponin drugs, congealing point of volatile
and fixed oils, melting and boiling points and water contents are some important parameters
used in the evaluation of drugs.

Ultraviolet light is also used for determining the fluorescence of extracts of some drugs.

Moisture Content:

Presence of moisture in a crude drug can lead to its deterioration due to either activation of
certain enzymes or growth of microbes. Moisture content was determined by heating the bark at
150⁰C in an oven to a constant weight and calculating the loss of weight.

Agar well diffusion method:

The agar plate surface was inoculated by spreading a volume of the microbial inoculum over the
entire agar surface. A hole was punched with a diameter of 6 to 8 mm aseptically with a sterile
cork borer or a tip, and a volume (20–100 µL) of the antimicrobial agent or extract solution at
desired concentration into the well was introduced. The agar plates were incubated under
suitable conditions depending upon the test microorganism. The antimicrobial agent diffused in
the agar medium and inhibited the growth of the microbial strain tested

Microorganis Incubation Incubation

Methods Growth medium Final inoculum size
m temperature (°C) time (h)
Disk-diffusion (0.5 McFarland) (1–
Bacteria MHA 35±2 16–18
method 2)×108 CFU/mL
(0.5 McFarland) (1–
Yeast MHA+GMBa 35±2 20–24
5)×106 CFU/mL
Molds Non- (0.4–5)×106CFU/mL – –
supplemented

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Microorganis Incubation Incubation

Methods Growth medium Final inoculum size
m temperature (°C) time (h)
MHA
Bacteria MHB 5×105 ×CFU/mL 35±2 20
Broth Yeast RPMI 1640b (0.5–2.5)×103 CFU/mL 35 24–48
microdilution
48 for most
Molds RPMI 1640b (0.4–5)×104 CFU/mL 35
fungi
Bacteria MHB 5×105 CFU/mL 35±2 20
Broth Yeast RPMI 1640b (0.5–2.5)×103 CFU/mL 35 46–50
macrodilution
48 for most
Molds RPMI 1640b (0.4–5)×104 CFU/mL 35
fungi
Agar dilution Bacteria MHA 104 CFU/spot 35±2 16–20
0, 4, 18, and
Time-kill test Bacteria MHB 5×105 CFU/mL 35±2
24

MHA: Mueller Hinton Agar. MHB: Mueller Hinton Broth.

GMB: the medium was supplemented with 2% glucose and 0.5 mg/mL methylene blue.

Determination of total activity (TA)

Total Activity is the volume at which test extract can be diluted with the ability to kill
microorganisms. It was calculated by dividing the amount of extract from 1 g plant material by
the MIC of the same extract or compound isolated and was expressed in ml/g.

Determination of ash values

Total ash

Four grams of the powdered material was accurately weighed and placed in a previously ignited
and tared silica crucible. The material was spread in an even layer and ignited by gradually
increasing the heat to a temperature of 500–600°C until it was white, indicating the absence of
carbon. The material was cooled in a desiccator and weighed. The content of total ash was
calculated in mg/g of air-dried material.

Acid-insoluble ash

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To the crucible containing the total ash, 25 mL of hydrochloric acid was added, covered with a
watch glass and boiled gently for 5 min. The watch glass was rinsed with 5 mL of hot water and
this liquid was added to the crucible. The insoluble matter was collected on an ash less filter
paper and washed with hot water until the filtrate was neutral. The insoluble matter left on the
filter paper was transferred to the original crucible, dried on a hot plate and ignited to constant
weight. The residue was allowed to cool in a suitable desiccator for 30 min, and then weighed
without delay. The content of acid-insoluble ash was calculated in mg/g of air-dried material.

Extractive values

The extractive values were recorded in alcohol and water with a view to study the distribution
of various constituents of Dalbergiella nyasae. Accurately weighed 4.0 g of coarsely powdered
air-dried material was placed in a glass-stoppered conical flask and macerated with 100 mL of
the solvent for 6 h, shaking frequently, and then allowed to stand for 18 h. The mixture was
filtered rapidly taking care not to lose any solvent. Twenty-five millilitres of the filtrate were
transferred to a tarred flat-bottomed dish and evaporated to dryness on a water bath. The residue
was dried at 105°C for 6 h, cooled in a desiccator for 30 min, and weighed without delay.

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CHAPTER 5
RESULTS, DISCUSSION AND CONCLUSION
Antimicrobial activity of Dalbergiella nyasae
EXTRACT S. aureus P. aeruginosa C. albicans
IZ(mm) AI IZ(mm) AI IZ(mm) AI
Acetone 10 0.36 ± 0.006 15 0.48 ± 0.06 9 0.43 ±
0.12
Methanol 15 0.58 ± 0.13 14 0.54 ± 0.27 15 0.47 ±
chloroform(1:1) 0.13

STANDARDS

fluconazole IZ=20-35mm
amoxicillin IZ=15-30mm
IZ= Inhibition zone in mm (mean value; including 6mm diameter of hole/disc), AI= Activity index (IZ
developed by standard), ± = SEM, Extracts assayed in triplicate

MIC Values of Dalbergiella nyasae bark

EXTRACT S. aureus P. aeruginosa C. albicans

*
MIC *MIC *MIC
ACETONE 0.3125 0.3125 0.625
Methanol chloroform 0.3125 0.625 0.625
(1:1)
*in mg/ml

MIC= Minimum Inhibitory Concentration

Total activity of crude extracts of Dalbergiella nyasae

extract Total Activity (ml/g)

S.aureus P.aeruginosa C.albicans
acetone 58.9 58.9 29.4

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Methanol 78.08 39.04 95.36

chloroform(1:1)
extract per gram dried plant part
Total Activity=
MIC of extract

Antimicrobial activity (assessed in terms of inhibition zone and activity index) of the plant
extracts tested against different microorganisms were recorded. In the present study, 6 crude
extracts of acetone and chloroform methanol (1:1) were tested for their bioactivity. 4 crude
extracts showed some significant inhibitions against the test microorganisms. In this
investigation, the extracts showed strong bioactivity for crude extracts and the most susceptible
microorganisms were P. aeruginosa and S. aureus. Excellent antibacterial activity against S.
aureus were observed in chloroform methanol fraction of bark extract (IZ 15 mm, 0.58 ± 0.13,
MIC 0.3125 mg/ml) and acetone extract (IZ 10 mm, 0.36 ± 0.06, MIC 0,3125 mg/ml). The
excellent antifungal activity against C. albicans was also observed in acetone extract (IZ 9 mm,
0.43 ± 0.14, MIC 0.625 mg/ml). In the bark, best antibacterial activity against P. aeruginosa
were observed in methanol chloroform extract (IZ 14 mm, 0.54 ± 0.27, MIC 0.625 mg/ml) and
in acetone extract (IZ 15 mm, 0.48 ± 0.06, MIC 0.3125 mg/ml). The C. albicans possessed the
least bioactivity against the test microorganisms, in acetone extract (IZ 9 mm, 0.43 ± 0.12, and
MIC 0,625 mg/ml); in chloroform methanol extract (IZ 15 mm, 0.47 ± 0.06, MIC 0,625 mg/ml)

The range of MIC of extract evaluated for bioactivity in diffusion assay recorded were 0.31–5
mg/ml and 0.3125–10 mg/ml and 0.625–10 mg/ml and 0.625–2.5 mg/ml for, S. aureus; P.
aeruginosa and C. albicans respectively. In the present investigation, lowest MIC value 0.3125
mg/ml were recorded Against Gram negative P. aeruginosa and Gram positive S. aureus was
also recorded, showing significant antimicrobial potential of test extracts.
Amount of extracts isolated from the bark and total activity (TA) were calculated and recorded
in Table. 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 and chloroform methanol
crude extracts were 58,9ml/g and 95.36 ml/g against P. aeruginosa and C. albicans
respectively. The best solvent in extracting compounds was chloroform methanol (78, 08 mg/g)
and acetone (95,36mg/g). Most of the extracts recorded slightly high total activity (TA) values
against the test organisms even at low concentration indicating the potential to inhibit the
growth of microorganisms.

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The chloroform methanol had the greatest quantity of the material from the crude acetone
extract with a percentage yield of 49% while acetone had least materials with a yield of 11%.
The recovery of the fractions from the original crude extract was 67%. The qualitative
phytochemical screening of bark extracts showed the presence of alkaloids, flavonoids,
saponins, terpenoids, steroids, tannins and phenolic compounds. Alkaloids, flavonoids, saponins
were more abundant this confirms the presence of the phytoconstituents responsible for
antimicrobial activity in the bark extracts.

The values of percentage loss on drying were 8.25 ± 0.582, which indicates that the moisture
content of the Dalbergiella nyasae is within the range and depicts good flow characteristics.
The total ash, acid-insoluble ash, and water-soluble ash were found to be 19.146 ± 0.237, 2.351
± 0.223, and 49.216 ± 0.634, respectively. The value of total ash indicates that the inorganic
contents of the formulation are below the required limits. Extracts of the Dalbergiella nyasae
were prepared and evaluated for phytochemical analysis and extractive values, the results show
that alkaloids of the formulation are more soluble in chloroform methanol than in acetone and
the higher aqueous extractive value (45.784 ± 0.876) of the extracts depicts that chloroform
methanol is a better solvent of extraction for the formulation than ethanol.

DISCUSSION
Results of the present study showed that chloroform methanol (1:1) and acetone extracts from
root bark tested inhibited the growth of selected bacteria and fungi, indicating the broad-
spectrum bioactive nature of the D. nyasae herbal plant. C. albicans was the most susceptible
organism in the investigation after gram positive S. aureus and then lastly gram-negative P.
aeruginosa. These organisms cause infections that are very difficult to combat and they were
found to show susceptibility to the acetone and chloroform methanol (1:1) of the root bark
extracts indicating that the extracts had a bacteriostatic as well as fungistatic effect on the tested
microorganisms.

Susceptibility difference between the two bacteria, Gram positive S. aureus and Gram-negative
P. aeruginosa may be due to cell wall structural differences between them. The Gram-negative
bacterial cell wall outer membrane appears to act as a barrier to many substances. The

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qualitative phytochemical screening of the leaves and root bark extracts showed different
phytoconstituents composition of alkaloids, flavonoids, saponins, terpenoids, steroids, quinines,
tannins and phenolic compounds. 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
the root bark of D. nyasae therefore showing that the extracts are very effective against a wide
array of microorganisms. Their activity is probably 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 complex with sterols in the plasma membrane, thus
destroying the cellular semi-permeability and leading to death of the cell or microbes. 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 leakage from microbial cell or the exit of critical molecules and ions will lead to
death of microbial. 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 activities observed with the acetone and chloroform
methanol (1:1) extracts suggest 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 barks extracts displayed potent and relevant pharmacological activities
with antibacterial and antifungal activities against gram positive and gram-negative selected
bacteria. This study showed that Dalbergiella nyasae bark extract has pharmacological interest
for therapeutic application as antibacterial and antifungal activities against infectious diseases.
The results obtained showed that Dalbergiella nyasae could be of pharmaceutical interest for
therapeutic application as a complement in antibacterial and antifungal agents. The
chromatographic processes were further used for identification of phytoconstituents understudy.

The qualitative phytochemical screening of Dalbergiella nyasae stem bark showed different
phytoconstituents. The extract indicated that it had a bacteriostatic and fungistatic effects on
tested different microorganisms. The research also provides new scientific knowledge about
Dalbergiella nyasae stem bark based on its antimicrobial potential and phytochemical profiling

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which has not been reported. More work is recommended on crude extracts and fractionation of
bioactive compounds to show its structures and indicate the exact potential. To block many
pathogenic microbes and development of broad spectrum antimicrobial herbal formulation.
Further evaluation of cytotoxic effects of compounds and extracts need to be undertaken to
justify their use by traditional healers to treat various diseases.

APPENDIX
Identification of the plant done at the National Herbarium and Botanic Garden

Ministry of lands, agriculture, water and rural resettlement

DEPARTMENT OF RESEARCH & SPECIALIST SERVICES (DR&SS)

All Communications to be addressed to NATIONAL HERBARIUM &
“THE HEAD”
BOTANIC GARDEN
Telephone: 263-4-744 170, 745230
Box A889
E-mail: nhbg@drss.gov.zw

10 March 2020

Ms T. Mhlanga
Harare Institute of Technology

Dear Madam

RE: Plant Identification

The plant specimen you brought in for identification was identified as Dalbergiell1a nyasae.

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Thank you.

Man’ombe Z. (Ms)
Research Officer
National Herbarium and
Botanic Garden

GALLERY

Debarking the tree

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The process of extraction

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The process of testing

phytochemical constituents

The process of determining

the antimicrobial activity
of the extract

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