Microbiological Quality of Herbal Formulation used for the treatmentof Typhoid Fever Sold in Makurdi Metropolis, Central Nigeria
It is estimated that approximately80% of the population in
developing countries uses herbal medicine as part of their
primary health care. The use of herbs dates back to the time of
the early man, who had the crudest tools as his implements and
used stones to start his ire [1]. Medicinal science came into
Volume 2, Issue 1 2023 © 2023 Acta Traditional Medicine. All Rights Reserved.
Corresponding Author: Odo, J.I | E-Mail: (odojoel@gmail.com)
Received 01 January 2023 | Revised 20 March 2023 | Accepted 15 April 2023 | Available Online May 6 2023
ABSTRACT
existence through a Greek man called Hippocrates and as such
he earned his reputation as the father of medicine (Heber,
2008). Most savage people believed that disease was due to the
presence of evil spirits in the body and could be driven out only
by the use of poisonous and disagreeable substances calculated
to make the body an unpleasant place to remain. The
knowledge regarding the source and use of the various
products suitable for the purpose was usually restricted to
medicine men of the tribe [2].
Contamination of herbal medicinal products is deined as the
“undesired introduction of impurities of a chemical or
microbial nature or of foreign matter, into or onto a starting
material, intermediate product or inished product or inished
lavored product throughout production, sampling, packaging,
or repackaging, storage or transport”
The introduction of micro-organisms in herbal medicinal
products can alter the physicochemical characteristics of the
products, which may lead to harmful effects on the quality of
the herbal medicine products. The organisms he said were
Staphylococcus aureus, Escherichia coli, Rhizopus stolonifer,
Candida species, etc [3]. Bacteria are ubiquitous in nature,
therefore because of the ubiquitous nature of bacteria, they
contaminate almost every material, herbal juice no exception
[4].
Among the main safety risks related to medicine is
contamination by micro-organisms of various kinds that may
be adherent in leaves, stems, lowers, seeds, and roots from
which herbal medicines are prepared. Alternatively,
microorganisms can be introduced during harvesting,
handling, open-air drying, preserving, and manufacturing.
Although medicinal plants with their chemical constituents
carry huge applications in the treatment or prevention of
various diseases. The plant materials are exposed to various
contaminants like toxic elements, pesticides, residues, etc. but
the chief contaminants mainly responsible for the
deterioration of herbal products are the microbe. Several
studies showed that herbal plants are associated with broad
variety of microbial contaminants [5]. When herbal products
contaminated with these micro-organisms are consumed by
people, they can cause serious health issues, one of the most
serious issues surrounding the safety of herbal formulations
Odo, J.I., Acta Traditional Medicine (2023)
concerns regulating the level of mycotoxins found in herbal
preparations. Mycotoxins are a group of around 400 toxic
secondary metabolites produced by fungi such as Aspergillus,
Penicillium, Fusarium, Claviceps and Alternaria Recently [6]
showed that 22% of herbal plant samples studied failed to
comply with the quality requirement for traditional medicine.
Herbal medicine is most often polyherbal, being prepared from
mixtures of different plant parts obtained from various plant
species and families and may contain multiple bioactive
constituents that could be dificult to characterize [7]. The
bioactive principles in most herbal preparations are not always
known and there could be possibilities of interaction with each
other in solution. The quality as well as the safety criteria for
herbal drugs may be based, therefore, on a clear scientiic
deinition of the raw materials used for such preparations. Also,
herbal medicine may have multiple physiological activities and
could be used in the treatment of a variety of disease conditions
[8]. It could be administered in most disease states over a long
period of time without proper dosage monitoring and
consideration of toxic effects that might result from such
prolonged usage [9].
The usages of herbs as medicines are determined mostly by the
community and environment in which one grows up. [10]
carried out a study to determine the socio-demographic
characteristics and pattern of use of herbal medicines by
women admitted to the Obstetrics and Gynaecology
Department in the Komfo Anokye Teaching Hospital (KATH), a
teaching hospital serving the Northern part of Ghana and made
the following observations: More than ifty percent (50%) of
patients used herbal medicines which were mostly unknown to
the attending health workers. The less educated as well as the
unskilled/ semi-skilled used herbal medicines more frequently
compared to their more skilled and educated counterparts.
Herbal medicine use is thus more prevalent in the groups who
usually have poor socio-economic facilities and carry most of
the burden of social deprivation. It is possible that their disease
conditions may be adversely affected.
To achieve the desired beneit from herbal preparations, an
individual must take the required dose over a certain length of
time. Although it is generally believed that most herbal
preparations are safe for consumption, some herbs like most
biologically active substances could be toxic with undesirable
side effects [11].
- © 2023 Acta Traditional Medicine. All Rights Reserved.
MATERIALS AND METHOD
Study Area
The study was carried out in Makurdi, Benue State. It has a
population of 226,198 (1991 census) with a landmass of 16km
radius. It has major markets like the North Bank market,
Wurukum market, High-level market, Modern market, and
Wadata market.
Sample Collected
The commercially sold herbal formulation was purchased from
four different markets (North bank market, Wurukum market,
Modern market, and Wadata market). Two samples were
purchased from each market and were immediately
transported to the Department of Microbiology laboratory of
the Federal University of Agriculture, Makurdi for
microbiological analysis.
PREPARATION OF CULTURE MEDIA
Nutrient Agar
This is an all-purpose medium, very rich for the support of
bacterial growth, 28 g of Nutrient agar was weighted and
dissolved in 1000 ml of de-ionized water and autoclaved, it was
allowed to cool before it was aseptically poured into sterile
Petri dishes.
Potato dextrose agar (PDA)
65g of potato dextrose agar was dissolved in 1000 ml of deionized water, the powder was allowed to dissolve and the tube
was plugged with cotton wool, covered with aluminum foil, and
autoclaved at 121C for 15 minutes. It was allowed to cool
aseptically and poured into a sterile plate. The surface was
lamed to remove air bubbles. The plates were allowed to
solidify and then dried in the hot air dryer 10% lactic acid
(0.5ml) was incorporated to prevent the growth of bacteria and
other organisms apart from fungi.
Isolation of Microorganisms
One milliliter (1ml) of each sample was measured into a sterile
test tube containing 9ml of sterilized distilled water. The 10-
1suspension was serially diluted using tenfold serial dilution
up to 10-4. Aliquot of 1ml of the appropriate dilution was plated
in nutrient agar for the isolation of bacteria while Potato
dextrose agar was used for fungi isolation. The inoculated
nutrient agar plates were incubated at 37oC for 24-48 hours,
while PDA plates were incubated at room temperature (280C)
for 3-5 days. After incubation, the number of discrete colonies
was counted in terms of colony-forming units. The viable
counts were obtained by reference to the serial dilution used.
MICROBIOLOGICAL EVALUATION OF SAMPLE
Culture of samples
Each of the herbal formulation was shaken vigorously and
inoculated into two different media: Nutrient agar for the
cultivation of microbes, and PDA for the isolation and
cultivation of pathogenic fungi and yeast.
The liquid herbal formulation was diluted by dissolving 1g of
the sample in 9 ml of normal saline, the aliquots (0.1ml) of the
herbal preparations were inoculated into plates containing
different culture agar and incubated at 37ºC for 24-48 hrs for
visible colonies. The plates containing potato dextrose agar
were incubated at room temperature for 5 days. After
incubation, visible colonies that developed were enumerated
and recorded as colony-forming units/ml (cfu/ml) of cfu/g.
Identiication of colonies on the different media
The following parameters were used to identify colonies on
Nutrient Agar and potatoes dextrose Agar. The different
parameters used include; Colour, Elevation, opacity, size, and
surface.
Subculture
This was done in order to obtain a pure culture for the
identiication of organisms, A discrete colony of the organism to
be identiied was collected from cultured plates, using a sterile
wire loop and subcultured on mannitol salt agar, salmonella
shigella agar, EMBA, using a streaking method and incubated
for 24 hrs at 37C for bacteria.
Again a discrete colony from the fungi culture was sub cultured
into another PDA and allowed to grow for 72 hours, after which
identiication of the organisms were done.
Odo, J.I., Acta Traditional Medicine (2023)
served during consumption of the crop, medicinal uses of the
crop ruminant ownership of the farmers and their fodder uses
of breadfruit parts. - © 2023 Acta Traditional Medicine. All Rights Reserved.
2.3 Sampling Method
Citrate Utilization Test
CHARACTERIZATION OF ISOLATES
Gram Staining
This is a differential staining that classiies bacteria into grampositive or negative. Thin smears of fresh, pure bacteria
cultures were made on clean grease free glass slides. The slides
were allowed to air dry and heat-ixed by passing them over a
Bunsen burner lame. The slides were placed on a rack over a
sink and looded with crystal violet for 60 seconds, the slides
were rinsed with clean running tap water and looded with
iodine for 60 seconds, the slides were rinsed with water and
decolorized with 95% alcohol for 30 seconds, the slides were
counter stain with safranin 60 seconds, they were rinsed with
clean running water and allowed to air dry after which they are
viewed under the microscope using oil immersion lens
(100x).Gram-positive bacteria stained purple while gramnegative bacteria stained red.
BIOCHEMICAL TEST FOR IDENTIFICATION OF
BACTERIA
Indole Test
This test is carried out to test the ability of an organism to break
down tryptophan, an amino acid to pyruvate and indole. The
test organism is inoculated into 5 ml of sterile peptone water
and incubated aerobically at 37C for 24 hrs. The production of
indole was tested by adding 0.5 ml of Kovac’sreagent to the
broth culture and shaken gently. A positive result shows a red
colour in the alcohol layer which indicates that indole was
produced while a negative result shows the absence of red
colour.
Catalase Test
The test demonstrates the presence of catalase which is an
enzyme that catalyzes the release of oxygen from hydrogen
peroxide (H2O2). A colony of 24 hours old culture was picked
using a sterile loop and then emulsiied in a few drops of
hydrogen peroxide on a clean slide. The presence of
effervescence indicated catalase positive reaction where as a
negative reaction showed no effervescence
Coagulase Test
The slide method described by [11] was used for the test. A drop
of normal saline was placed on each end of a slide. An 18-24
hours old culture of the test organism was emulsiied in each of
the drops to make two thick suspensions. Thereafter, a drop of
human plasma was added to one of the suspensions. The
mixture was stirred for about 5 seconds. Clumping of the
organism within 10 seconds is a positive test. No plasma was
added to the second suspension which is the control to
differentiate any granular appearance of the organism from
true coagulase clumping.
Urease Test
Isolates were inoculated into liquid urea agar supplemented
with urea and aseptically dispensed into sterile test tubes, and
slanted to solidify. They were incubated at 370C for 24-48
hours. The development of bright pink or red color indicates a
positive urea reaction [12].
Simmon‟s citrate agar was prepared based on the
manufacturer’s instruction, sterilized, poured, and allowed to
solidify at a slant angle of 450C. The test organism was streaked
on the surface of the agar slant and then incubated at 370C for
48 hours. A change in the color of the agar medium from green
to blue following the growth of the organism on the slant
indicated a positive test, while no color change indicated a
negative test.
Fungal Identiication
The fungal isolates were identiied microscopically using
lactophenol cotton blue test. The identiication was achieved by
placing a drop of the stain on the clean slide with the aid of a
wire loop. A small portion of mycelium from the fungal culture
was removed and placed in a drop of lactophenol. The
mycelium was spread on a slide with the aid of the wire loop. A
cover slip was gently applied with little pressure to eliminate
air bubbles. The slide was then mounted and observed with x10
and x40 objective lenses respectively.
RESULTS
Table 1 Presents the Morphological and bacterial counts of
each sample in 4 different markets in Makurdi metropolis. It
shows that MM1 (Modern market 1) had the highest counts of
12.5x103cfu/ml, while WD2 (Wadata 2) had the least count of
1.7×103.
Table 1: Morphological and colony count of bacteria isolated
from herbal formulation from 4 different markets in Makurdi
metropolis.
Table 2 Shows the average counts of fungal isolates which
shows that NB1 (North bank 1) had the highest count of
2.5×103 cfu/ml, while the least count was obtained from WD1
(Wadata 1) with 1.0×103 cfu/ml.
Table 2:Average colony count of fungal isolates from the herbal
formulation in 4 different markets in makurdi metropolis
Table 3 and 4 show the occurrence of bacterial isolates from
the sample collected from four different markets.
Staphylococcus aureus had the highest frequency of 37.5% of
the isolates in the sample. Bacillus spp had the lowest frequency
of 12.5% of the bacterial isolates in the sample.
(North Bank 1) and WK2 (Wurukum 2) had all the bacterial
isolates recorded in this study from herbal formulation, while
WD1 (Wadata 1) had only 1 bacterial isolate.
Odo, J.I., Acta Traditional Medicine (2023) - © 2023 Acta Traditional Medicine. All Rights Reserved.
consumption, there is a very high chance of passing the
antibiotics resistant microorganisms into the human
ecosystem. This poses a great danger to human health. Since
herbal concoctions are prepared using varieties of medicinal
plants that contain active constituents that are cheap and
effective against common bacterial infections.
A number of sources of contamination of herbal preparation
especially during preparation have been identiied. The
microlora of the inal product may represent contaminants
from the raw materials, equipment, water, and atmosphere and
from personnel. Microorganisms such as Escherichia coli, and
Scedosporiumspp reported in this study are generally known to
proliferate in potable, distilled, and de-ionized water while
Bacillus spp, Staphylococcus, Aspergillus and penicillin are
commonly isolated from air. The most common source of postpreparation herbal contamination is the packaging vessels. In
order to enhance consumer acceptability, most herbalists in
Nigeria have adopted the use of bottles and plastic containers
as packaging vessels for their preparations. Unfortunately,
these vessels are not subjected to any form of sterilization after
washing them. Contamination of the preparation coupled with
the humid tropical environments may result in the proliferation
of microbial contaminants in the herbal remedies (WHO,
2007); this probably explains the high microbial counts
recorded in this study. Such high levels of microbial
contamination have been shown to result in spoilage and
degradation of the products or may constitute a health hazard
to the user [14]. Most herbal preparations are made up of
different components of various plant species and these
preparations are not standardized with respect to color, taste,
consistency, etc. Unlike orthodox drugs, changes in the
appearance, odor, taste, etc of herbal formulations due to
spoilage are hardly readily detected by the patients [13].
Among the microorganisms isolated from Herbal Medicine,
Bacillus, Staphylococcus, E. coli, and penicillin were the major
contaminants. Although the pathogenicity of these organisms
was not assessed, species of these agents have been
incriminated in serious human infections. Bacillus spp are
widely distributed in the soil, dust, air, and water and they are
resistant to environmentally destructive factors. Apart from the
unacceptable microbial loads observed in the samples, the
presence of contaminants considered to be completely
unacceptable in herbal preparations was demonstrated. The
most common isolates in the tested samples were Grampositive organisms belonging to the genera Bacillus and
Staphylococcus aureus are normal commensals of the
mammalian skin, hands, and mucous membranes. Upon
consideration of the extent of human contact involved in the
preparation of herbal medicinal samples, it is most likely that
sources of the contaminating Staphylococcus spp. are the
producers of the herbal formulations this suggests that the
level of hygiene of persons involved in the preparation of the
tested samples may be low. Similar studies carried out on
herbal samples include work by [15-20] have all reported that
the pathogens frequently isolated in herbal products were S.
aureus, E. coli. Except for Scedosporium which was not isolated
in some reports mentioned above. This work varies by
reporting a higher count of Bacillus spp. Contamination by
Bacillus spp could have arisen during growth of the herbs as the
bacterium is commonly found in soils. Escherichia coli, a major
faecal coliform may have been introduced from the water used
during the processing of the herbs. Microbial contamination of
Table 3: Occurrence of bacterial isolates from the herbal
formulation in 4 different markets in makurdi metropolis
Table 4: Occurrence of the bacterial isolates from the different
markets
- = Absent + = Present
The percentage occurrence of fungal isolates from the herbal
formulation,shows that Scedoporiumsppand Penicillium spp
both had the same frequencies of occurrence representing
37.5% of the total fungal isolates. While Aspergillus spp, had a
percentage frequency 12.5% of the total fungal identiied.
(Table 5)
Table 5: Percentage occurrence of fungal isolates from the
herbal formulation in 4 different markets in Makurdi
metropolis
On table 6 the occurrence in percentage of fungal isolates in
four different markets is presented. The study shows that WK2
(Wururkum 2) had all the fungal isolates in the herbal
formulation identiied representing the highest frequency of
occurence, while Mm1, (Modern market 1) had had the least
fungal isolates.
Table 6: Occurrence of fungal isolates from the herbal
formulation in 4 different markets in Makurdi metropolis - = Absent + = Present
DISCUSSION
Thi s s tudy has shown that there are var ieties of
microorganisms present in our various herbal formulations
which could have resulted from contaminated soils, plants and
its products, preparation processes, quality of water,
containers, and processing equipment. However, these
microorganisms exhibit multi-resistance to many antibiotics.
Since herbal formulations are mainly prepared for human
Odo, J.I., Acta Traditional Medicine (2023)
herbal mixture as shown in this study, may also be a result of the
plant materials, utensils used during preparation, poor hygiene
of the manufacturer, or even the packaging vessel after
processing as also suggested by many authors earlier reported.
Microorganisms are present everywhere and can easily
contaminate any substrate. Considering the packaging
materials, it is worthy of note that this contributes greatly to
microbial contamination as the inal stage of the processing is
the packaging. Most of the packaging cans used are not
sterilized and are usually picked up where they are found
littered along the road or in public places and barely washed
before being used to package inished herbal products. It was
observed that fungal growth was more than bacterial growth
and this is attributable to the low pH value of all the herbal
formulation samples which is favorable for fungal growth. The
high microbial load and presence of speciic pathogens in the
tested herbal formulation heave serious clinical as well as
pharmaceutical implications. Clinically, consumers of any of
these products are at risk of contracting infections by the
different pathogens which may be of great consequence if not
identiied and treated appropriately. The presence of
Escherichia coli in the sample indicates poor hygiene practices
and a lack of adequate handling of the products. According to
the European pharmacopoeia 2007, no Salmonella spp or
Escherichia coli strain should be present in samples [21]. The
intake of a high concentration of accumulated toxins produced
by organisms such as Bacillus cereus, Staphylococcus aureus,
and Aspergillus niger may lead to undesirable reactions in
consumers. [22] reported that the presence of Bacillus spp. in
herbal preparations is an indication that the water used in the
preparation of the products is not from a good source. The risk
is greater if the consumer is a young child with undeveloped
immunity, an elderly with diminished immunity, or the
immunocompromised patients. Incidentally, these groups of
consumers are the most in need of herbal medicines for the
treatment of many diseases to which they are susceptible
- © 2023 Acta Traditional Medicine. All Rights Reserved.
CONCLUSION
Staphyococcus aureus, Escherichia coli, Enterobacter spp,
Bacillus spp Scedosporidum spp Penicillium spp, Aspergillus
niger, and Phialophoraparasiticumsppwere isolated and
identiied as micro-organisms associated with contamination
of herbal formulations sold in markets within Makurdi
metropolis, with Staphylococcus aureus having the highest
bacterial colony count and Scedosporium spp having the
highest fungal count.
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