PROJECT PROPOSAL ON EXAMINATION OF THE MICROBIAL QUALITY AND

THE ANTIBACTERIAL PROPERTIES AND SENSITIVITY PATTERN OF HERBAL

PRODUCTS PRODUCED IN NIGERIA

Introduction
The use of herbal medicine has always been part of human culture, as some plants possess
important therapeutic properties, which can be used to cure human and other animal
diseases. Rios and Receo (2005) reported that the idea that certain plants had healing potentials,
indeed, that they contain what would be characterized as antimicrobial principles was well
accepted long before mankind discovered the existence of microbes. The healing property of
these medicinal plants is usually linked with the presence of secondary metabolites and these
differ from one plant to another. It has been reported that a substantial percentage (38%) of
prescription contained one or more of the natural products of plant origin as the therapeutic agent
(Farnsworth and Bringel, 2017).
The use of plant, plant extract or chemical derived from plants to treat disease is therapeutic
modalities which has stood the test of time (Anwannil and Atta, 2005). Suck (2019) had earlier
reported that more than 75 pure compounds derived from higher plants are used in herbal
medicine but most of those applied in modern medicine are now produced synthetically. In
recent studies, extract of various parts of medicinal plants were found to have broad spectrum
antimicrobial activities against pathogenic organisms (Sudhakar et al., 2006; Khan et al.,
2006; Oyetayo and Oyetayo, 2006).
Herbal medicine is more accessible to most of the population. About 60 to 85% of the
populations of every country of the developing world rely on herbal or indigenous forms of
medicine. The reasons for the high patronage of herbal medicine are the high cost of very
effective antibiotics and the problem of antibiotic resistance which is very common in
developing countries (Hack, 2005; Okeke et al., 2019).

Statement of the Problem

A record of medicinal plants in earliest period in Nigeria is virtually not available because there
was no documentation for their isolation, selection and preparation. Every fact about potent
herbal plant was passed by word of mouth from generation to generation (Kochhar, 1981). In the

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 last two decades, there has been an upsurge in the circulation of herbal products in Nigeria. Most
of the product developers often have bogus claims that their products can cure all forms of
ailments. A greater number of residents in Kaduna metropolis are believed to depend on
traditional herbalists for their medical needs. Unfortunately, no researches (to the best of our
knowledge) have been carried out to determine the microbiological safety of these herbal
products in Kaduna metropolis. In this paper, the level of contamination of powdered herbal
products marketed in Kaduna metropolis with selected pathogenic bacteria and the susceptibility
of these contaminants to commonly prescribed antibiotics in the metropolis are reported.

Aim of the Study

The present study was therefore meant to examine the microbial quality and the antibacterial
properties and sensitivity pattern of herbal products produced in Nigeria.

Objective of the Study

The specific objective of the study are
1. Examine hawked herbal remedies
2. Determine the microbial quality of the herbal mixture.
3. Examine the sensitivity pattern of the herbal mixture.
4. Provide relevant recommendation due to the findings.
Justification of the Study
In Nigeria, due to the cultural acceptability of healers, the relatively low cost of traditional
medicine, and limited access to modern health facilities up to 80% of the population uses
traditional medicine (Sudhakar et al., 2006). Nigeria has high diversity of plant species most of
which are used in the preparation of herbal medicinal products and it is one of the six African
countries where about 60% of the plants are said to be indigenous with their healing potential
Okeke et al., 2019).The growing and unhygienic use of herbal medicinal products has become a
major concern in public health. However, in Nigeria particularly in the study area, there is no
sufficient data which addresses the bacteriological quality of herbal medicinal products. Hence
the aim of this study was to assess the occurrence of potential bacterial pathogens and their
antimicrobial susceptibility patterns isolated from herbal medicinal products sold in Nasarawa
(Okeke et al., 2019).

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.

Literature review

Herbal medicine (also Herbalism) is the study of pharmacognosy and the use of medicinal plants.

Plants have been the basis for medical treatments through most of human history, and
such traditional medicine is still widely practiced today(Sudhakar et al., 2006). Modern medicine
makes use of many plant-derived compounds as the basis for evidence-based pharmaceutical
drugs. Although herbalism may apply modern standards of effectiveness testing to herbs and
medicines derived from natural sources, few high-quality clinical trials and standards for purity
or dosage exist. The scope of herbal medicine is sometimes extended to
include fungal and bee products, as well as minerals, shells and certain animal parts (Anwannil
et al, 2016).

Herbal medicine is alsocalled phytomedicine or phytotherapy. Paraherbalism

describes alternative and pseudoscientific practices of using unrefined plant or animal extracts as
unproven medicines or health-promoting agents (Hack, 2016).  Paraherbalism differs from plant-
derived medicines in standard pharmacology because it does not isolate
or standardize biologically active compounds, but rather relies on the belief that preserving
various substances from a given source with less processing is safer or more effective – for
which there is no evidence. [4] Herbal dietary supplements most often fall under the phytotherapy
category.

METHODOLOGY
Source of herbal products
A total of 20 different herbal preparations will be purchased randomly from identified herbal
shops and retail outlets in different parts of Nasarawa metropolis. Packaged herbal samples will
be collected and taken to the laboratory, while those that will be not packaged (such as herbal
preparations sold by local herbalist) will be collected in sterile polythene bags (Pearce et al.,
2004). All samples collected from the sites will be analyzed in the laboratories of Department of
Microbiology.

Physical analyses

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Determination of pH
The pH of the products will be determined (Norris and Ribbons, 2017) by using Hanna
Microprocessor pH meter. Sample solution (10%) will be prepared by weighing 10g of the
sample into 200ml beaker and adding sterile distilled water (100ml) with shaking to obtain a
homogenous solution. There after the pH of the preparation will be taken with Hanna
microprocessor pH meter - pH 211 (Hanna Instruments, UK).

Determination of moisture content

A Motler Toledo HR73 Halogen moisture analyzer will be used to determine the moisture
content of the powdered herbal preparations. One gram (1g) will be weighed into a clean pan
(which will be a part of the machine accessories). The analysis will be done automatically and
the reading will be shown in percentage on the screen (NAFDAC SOP, 2000).
Bacteriological analyses
General purpose nutrient media, enrichment media and other appropriate selective media (all
obtained from Oxoid) will be employed in the culturing and isolation of selected pathogenic
bacteria in the study (Prescott et al., 1999).
Preparation of media
All dehydrated media will be prepared according to manufacturer's instructions. They will be
mixed with distilled water and dissolved by gentle heat to boil. The media will be sterilized in an
autoclave (LTE J7090 model, LTE Scientific Ltd, England) at 121°C for 15 minutes. The sterile
media will be dispensed or poured into sterilized Petri dishes and allowed to cool. The sterility of
the prepared media will be checked by incubation of blindly selected plates at 37°C for 24hrs.
Total aerobic bacterial plate count
The method of Colle and Miles (1989) will be used with some modifications. A stock solution of
the sample will be prepared by weighing one gram (1g) of the sample into 25ml of 0.1% sterile
peptone water and shaken thoroughly. A ten-fold serial dilution of the bacterial suspension will
be made. This will be done until 10−7 dilution will be achieved. 0.1ml will be then pipetted from
the 10−7 dilution onto the surface of each of two Petri dishes containing 20ml of a solidified and
sterile Plate Count Agar (PCA), and then spread evenly with sterile glass spreader. The plates
will be then incubated for a maximum of 48 hr at 37°C (including the control plates). Counting
of the bacterial colonies will be done using the Stuart Digital colony counter.

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Isolation and identification of Staphylococcus aureus
Enrichment of the bacteria will be done by adding one gram (1g) of the sample into peptone
water in sterile McCartney bottle and incubated for 18 hr at 37°C. Isolation of
the Staphylococcus aureus will be achieved by streaking the pre-enriched culture from the
peptone water on to a selective differential agar plate of Mannitol Salt Agar (MSA) which will
be freshly prepared following manufacturer's instructions. The plates will be then incubated at
37°C for 24 hr under aerobic conditions. Colonies showing golden yellow colour or colourless
will be considered to be Staphylococcus and will be subjected to biochemical tests such as
catalase as well as slide and tube coagulase for the confirmation of Staphylococcus aureus.
Isolation and identification of Escherichia coli
Enrichment of the bacteria will be achieved by adding one gram (1g) of the sample into a sterile
McCartney bottle containing sterile nutrient broth and incubated for 24 hr at 37°C. The pre-
enriched culture will be then inoculated into the selective differential E. coli medium and
incubated at 44°C for 24 hr. Inoculum from E. coli medium will be then streaked onto the
surface of an Eosin methylene blue (EMB) agar and incubated for 24 hr at 44°C. Dark colonies
with metallic sheen indicated the presence of lactose fermenters and will be confirmed to
be Escherichia coli by IMViC tests (Prescott et al., 1999).

Isolation and identification of Salmonella typhi

Enrichment will be done by transferring 1gm of the sample into 10ml of selenite cysteine broth
and incubated for 24 hr at 37°C. The enriched culture after incubation will be then streaked on
duplicate plates of freshly prepared Bismuth Sulphite Agar (BSA), (one lightly and one heavily),
and incubated alone with the control plate of Bismuth Sulphite Agar (BSA) at 37°C for 24 hr.
After incubation, typical black and green colonies will be regarded as positive for Salmonella.
The colonies will be then streaked on the nutrient agar slants for further biochemical
identifications using Triple Sugar Iron (TSI) and Lysine Iron Agar (LIA) tests.
Isolation and identification of Shigella spp
Enrichment of the bacteria will be achieved by introducing 10g of the sample to a prepared
sterile nutrient broth with pH adjusted to 8.0 and incubated for 18 hr at 37°C. The pre-enriched
culture will be then streaked onto the freshly prepared selective differential agar plates
of Salmonella-Shigella agar. Incubation will be done at 37°C for 48 hr. Non lactose fermenters

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 that grew as colourless colonies will be identified with suitable biochemical test such as Triple
Sugar Iron (TSI) agar test. Confirmation of Shigella isolates will be by serological tests.
Antibacterial susceptibility tests
To determine the spectrum of antibacterial activity of antibiotics (commonly sold in the study
area), the Clinical Laboratory and Standard Institute (CLSI, 2005) method will be adopted.
Preparation of paper disks
The dilution formula below will be used to prepare various concentrations of the antibiotics from
the stock solutions for the disk potencies (Taura et al., 2004).
The dilution formula: V1= R (V2/O) where;
 V1 = initial volume of the solution. V2 = final volume of the solution. R = required
concentration. O = observed concentration.
The prepared standard paper discs measuring 6mm in diameter will be sterilized in a clean bijou
bottle in an autoclave.
Preparation of antibiotic stock solutions
Standard solutions of antibiotics will be prepared by reconstitution of the powder as described by
the manufacturers to give a concentration of 125mg/5ml. Doubling dilutions will be carried out
with the final concentration in the tubes as follows: 25.000mg/ml, 12.500mg/ml, 6.250mg/ml,
3.125mg/ml, 1.563mg/ml and 0.782mg/ml. Disks (100) will be placed into each of the six tubes
with final concentration of 25.000mg/ml, 12.500mg/ml, 6.250mg/ml, 3.125mg/ml, 1.563mg/ml,
0.782mg/ml respectively until all the solution of antibiotics will be completely absorbed and the
disks properly impregnated with antibiotics. The impregnated disks will be then allowed to dry
in LTE IP30-UF oven for 20 mins at 30°C.

Test bacteria and antibiotics used for the susceptibility test

Bacteria isolated from the herbal preparations will be screened for their susceptibilities to the
selected antibiotics. They included: Escherichia coli, Staphylococcus aureus, Salmonella
typhi and Shigella spp. The antibiotics screened included: amoxicillin, ampicillin,
chloramphenicol, erythromycin, streptomycin, cefaclor, tetracycline and ofloxacin. The
antibiotics have also been recommended for routine dilution and disk diffusion susceptibility
tests (Yolker et al., 2005).

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Determination of zones of inhibitions using disks diffusion test
Clinical Laboratory and Standard Institute (CLSI, 2005) disk diffusion method will be employed.
To prepare the inocula, five colonies of bacteria will be picked from culture media and
inoculated into Mueller-Hinton broth and incubated at 37°C for 24 hr. Approximately 0.5ml of
the culture will be pipetted onto Mueller-Hinton agar surface, and spread evenly with a sterile
bent glass rod. A sterile pair of forceps will be used to place the appropriate antibiotic test disks
on the cultured plates. The plates will be then incubated at 37°C for 18 hr. The diameters of the
zones of inhibition will be measured (to the nearest mm) using a transparent meter rule.
Determination of Minimum Inhibitory Concentrations (MIC)
This will be determined using the broth dilution technique (Prescott et al., 1999). Decreasing
concentrations of the desired antibiotic will be challenged with standardized bacterial
suspension. The lowest concentration of antibiotics that inhibited cell growth, as validated on a
recovery broth (nutrient broth supplemented with yeast extract and tween 80), will be taken as
the MIC.
Expected Result
A total of 7 microorganisms will be isolated from the two herbal products. The distribution of the
isolates in the herbal products is shown in Table 1. A total of 4 fungi
(Basidiobotrytis sp, Oedocephalum sp, varicosporium sp and Articulospora inflata) and 3
bacteria (Bacillus subtilis, Bacillus coagulans and Bacillus cereus).