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ORIGINAL ARTICLE
Anti-shigellosis activity of the aqueous extract of garlic,
clove and fenugreek
Sara Adel Amer | Hanaa Sayed Mohamed Abd El-Rahman
Agricultural Research Centre (ARC), Food
Technology Research Institute (FTRI), Giza,
Egypt
Correspondence
Sara Adel Amer, Agricultural Research Centre
(ARC), Food Technology Research Institute
(FTRI), Giza, Egypt.
Email: dsaramer55@yahoo.com
Abstract
Shigellosis causes severe diarrhea and, in some cases, death in children. In this study,
we investigated the in vitro and in vivo anti-shigellosis efficacy of some aqueous
herbal extracts or their chosen mixtures, including fenugreek seed, black seed, clove,
garlic, and ginger. The antibacterial potential was assessed by well diffusion and mic-
rodilution assays. The extracts' minimum inhibitory concentrations ranged from 0.39
to 12.5 mg/ml. The water extract of garlic and combinations of garlic with either
fenugreek or clove extracts had the highest antibacterial efficacy, hence these
extracts were chosen for the in vivo investigation. Rats were given a Shigella
dysenteriae inoculum and then administrated two doses (100 and 200 mg/kg bw) of
the selected herbal extracts or the antibiotic ciprofloxacin (20 mg/kg bw) to treat
Shigellosis in vivo. The results showed that garlic and clove extract mixtures (G6,
200 mg/kg bw), garlic and fenugreek mixtures (G7 and G8, 100 and 200 mg/kg bw),
and ciprofloxacin group (G9, 20 mg/kg bw) had the best anti-Shigellosis efficacy. G7
(garlic and fenugreek mixtures (100 mg/kg bw) outperformed almost all other groups
evaluated. This group had lower defecation frequency, lower stool-Shigella loudness,
restored body weight, recovered hematological, and liver and kidney functions,
besides exhibited no anatomical alterations in the colon tissue that were comparable
to the uninfected group. As a result, in the treatment of Shigella, this extract combina-
tion may be suggested as a viable therapeutic alternative to antibiotics.
1|INTRODUCTION
A more dangerous form of gastroenteritis, shigellosis, also known as
bacillary dysentery, can cause extreme diarrhea and even death in chil-
dren (Khalil et al., 2018). It is regarded as a global health concern. Shigella
was indeed the second-leading source of diarrheal mortality in 2016
across all ages and make up nearly 13% of all diarrhea-related deaths
(WHO, 2020). Shigella dysentery affects approximately 270 million
individuals over the age of 5 years and between 28,000 and 64,000
incidences of death among children below 5 years annually (Lanata
et al., 2013). The major burden of Shigellosis is concentrated in low- and
middle-income countries. Disappointingly, there are currently no licensed
vaccinations against Shigella infection (Butkeviciute et al., 2021).
Shigella is a rod-shaped gram-negative, non-motile, non-spore-
forming bacteria that can cause shigellosis in humans. Ingestion of as
little as 100 Shigella germs results in severe diarrhea within 4–7 days.
When aggressive Shigella germs are swallowed, the bacterial infection
invades the terminal ileum, epithelial lining, rectum, and colon, as well
as the intestinal lining. Shigella symptoms include stomach pain,
watery diarrhea, or dysentery (several scanty, bloody, mucoid stools),
fever, vomiting, and dehydration (Al-Dahmoshi, Al-Khafaji, Al-Allak,
Salman, & Alabbasi, 2020). Thrombocytopenia, hemolytic uremia, and
kidney failure can develop if the condition is not treated properly (Ali
Nor, Menza, & Musyoki, 2021).
Unfortunately, some Shigella species are becoming increasingly
resistant to the most extensively used antimicrobials (WHO, 2018)
and in particular, the sd1 strain of S. dysenteriae which develops resis-
tance to the present antibiotic therapy rather quickly (Williams &
Berkley, 2018). For this reason, research has focused on using natural
remedies to combat bacterium resistance. The most common strategy
Received: 6 January 2022 Revised: 27 February 2022 Accepted: 21 March 2022
DOI: 10.1111/jfs.12978
J Food Saf. 2022;e12978. wileyonlinelibrary.com/journal/jfs © 2022 Wiley Periodicals LLC. 1of17
https://doi.org/10.1111/jfs.12978
was to test the antibacterial activity of plant extracts used in folk medi-
cine. These natural remedies are high in phytochemicals such as poly-
phenols and flavonoids, which are recognized as antibacterial agents
(Ríos & Recio, 2005). The majority of these are typically regarded as
food-safe ingredients with mild side effects (Nabavi et al., 2015). As a
result, these natural plants may hold promise for the development of
novel antimicrobial drugs against foodborne and human diseases.
Among the well-known natural plants that are used as spices and herbs
are clove, garlic, ginger, fenugreek, and black seeds.
Clove (Syzygium aromaticum) is a spice that may be found in prac-
tically all cuisines throughout the world. Clove comes from a tree
called Eugenia caryophyllata. It possesses many characteristics includ-
ing anti-oxidant, anti-insecticidal, antifungal, and antibacterial capabili-
ties. Traditional food preservation uses it as a flavor and antibacterial
agent (Parham et al., 2020). While ginger (Zingiber officinale), is classi-
fied as a member of the Zingiberaceae family (Mao et al., 2019). Fresh
and dried ginger roots were considered separate therapeutic medi-
cines in the ancient world. Colds, nausea, asthma, coughing, and colic
have all been treated with fresh ginger crude extracts. It has also
shown a good action against multidrug-resistant bacteria and rheuma-
tism (Karuppiah & Rajaram, 2012).
Other natural food additives include garlic (Allium sativum), which is
a part of the Alliaceae family of plants. It has been known for a long time
that garlic has antimicrobial properties (Johnson, Olaleye, &
Kolawole, 2016). On top of this, it is said to help in prevent heart dis-
ease (such as atherosclerosis), strengthen the immune system, and pro-
vide cancer prevention (Bayan, Koulivand, & Gorji, 2014). Because of its
therapeutic advantages, fenugreek (Trigonella foenum-graecum)haslong
been employed for ages. Research has discovered fenugreek's antiviral,
antibacterial and hypotensive characteristics, as well as its anti-cancer
and anti-inflammatory capabilities (Goyal, Gupta, & Chatterjee, 2016).
Whereas black seed (Nigella sativa) is an important miracle herb with an
extensive religious and historical background, it is being used to cure a
variety of ailments, such as parasitic infections, diarrhea, appetite stimu-
lation, and immune system support (Ahmad et al., 2013).
Several studies have shown that the aforementioned plants have
potent antibacterial and antioxidant properties due to their phyto-
chemical composition. As an example, euganol in clove can interact
with bacterium protein, causing component leaking. Allicin from garlic
works by attaching to the thiol groups of several bacterial enzymes.
Other phytochemicals, on the other hand, such as phenolic com-
pounds, flavonoids, alkaloids, saponin, and tannins, can act also as
antioxidants and protect cells from the destructive effects of reactive
oxygen species (ROS) such as superoxide anion, hydroxyl radical, and
hydrogen peroxide, and free radicals. These ROS play an important
part in the development of a variety of diseases, including immuno-
depression, diabetes, aging, dementia, cancer, and Parkinson's disease
(Parham et al., 2020).
Hence, our objective was to examine the antibacterial activity of
the aforementioned herbs against S. dysenteriae type I (sd1) in vitro, as
well as to assess the effectiveness of the powerful herbs in preventing
shigellosis in Shigella-infected rats, as demonstrated by hematological,
serological, and histological evaluations.
2|METHODS
2.1 |Preparation of the extracts
Fresh garlic and ginger were acquired from a local market, peeled and
cleaned, and 500 g of each washed with sterilized- distilled water
before being mixed individually with 100 ml of sterile distilled water
and filtered out using sterile cheesecloth. Then, 1 ml of the filtrate
was oven-dried at 40C until constant weight was achieved to deter-
mine its exact concentration. A working solution was made by dilution
to yield 200 mg/ml.
Before the extraction operation, fenugreek seeds, black seeds,
and cloves were pulverized in a laboratory mill in a 250 ml flask. Then
20 g of each dried spice was combined with 100 ml of boiled distilled
water to make a water extract. The mixture was rapidly agitated
before being left to stand for 24 h at 25 ± 5C. The mixture was then
filtered using sterile cheesecloth. Each obtained extract was then
transferred to pre-weighted glasses, which were then placed in an
oven set at 40–60C and the working solution (200 mg/ml) was made
as previously described. All crude extracts were filter-sterilized kept at
18C, and thawed before use.
The following herbal water extracts were prepared: fenugreek
seeds (WF), black seed (WB), clove (WC), garlic (WG), and ginger
(WZ). Mixtures based on a ratio of 1:1 were also developed from the
working solution of the herbal extracts, including garlic and glove mix-
tures (1:1) (WGC), garlic and fenugreek mixtures (1:1) (WGF), and gar-
lic, fenugreek, and clove mixtures (1:1:1) (WGFC).
2.2 |Antibacterial activity of extracts detection
using well diffusion methods
The antibacterial activity of the obtained extracts against
S. dysenteriae type I (sd1) was initially investigated using the well diffu-
sion technique (Toba, Samant, & Itoh, 1991). 18 h incubated bacterial
strain was inoculated in 100 ml of peptone water and its turbidity
level was matched against a 0.5 Mcfarland Standard to get a dilution
of 1.0 10
6
CFU/ml. Sterilized Muller Hinton agar (MHA, Biolife) was
poured into plates and surface seeded with the indicator strain. Wells
were formed on agar plates containing the indicator strain, and each
well was inoculated with 50 μl of each extract (100 mg/ml). Ciproflox-
acin discs 5 μg were also plated as a positive control, and after a 24 h
incubation period at 37C, an inhibitory zone broader than 6 mm in
diameter was measured.
2.3 |Determination of the minimum inhibitory
concentration of herbal water extracts by the
microdilution method
Minimum inhibition concentrations (MIC) of the crude water extracts
of each herb towards the examined S. dysenteriae type I (sd1) strain
were detected using the resazurin microdilution method (Elshikh
2of17 AMER AND ABD EL-RAHMAN
et al., 2016). A microtiter plate (96-well) enclosing 100 μl of double
concentrated Mueller-Hinton broth (MH, Biolife, Italiana) from col-
umns 1 to 9 were processed with serial twofold dilutions of the
stock extracts to make dilutions ranging from 100 to 0.39 mg/ml per
well. Whereby, columns 11 and 12 comprised 50 μlofMHbroth
media plus 50 μl of sterilized distilled water, and column 10 con-
tained 100 μl of the medium broth (as a blank to monitor sterility).
The standardized indicator strain suspension was adjusted to the
equivalent of 10
6
CFU/ml at OD 600 (CLSI, 2015). Then 10 μlofthe
prepared bacterial suspension was applied to all wells having the
extracts dilutions in addition to the control wells in columns 11 and
12, and then it was incubated for 24 h at 37C. Resazurin dye was
prepared (0.015%) and suspended to wells (30 μl per well), and incu-
bated again for 2–4 h to allow the color shift. After that, wells with a
blue resazurin color were considered the MIC (mg/ml). Along with
this, another microplate was used as a positive control and prepared
as previously described but with using Ciprofloxacin at the concen-
tration range of 0.078–10 μg/ml instead of extracts. For the calcula-
tion of MIC, the following formula was adopted: (A)2
b
,whereas
Arepresents the stock concentration (mg/ml) and bis the last well
showing blue color.
Whereas MBC was carried out by platting a 5 μl sample from MIC
wells on Mueller Hinton agar without extract. The MBC represents
the concentration at which there was no microbial growth. The
extracts tested in this study were screened three times against each
microorganism.
2.4 |Phytochemical screening
2.4.1 | Total polyphenols content (TPC) and total
flavonoids content (TFC)
The TPC was determined in the prepared extracts by adding 0.1 ml of
the Folin–Ciocalteu phenol reagent (1:1) and 1.5 ml of distilled water
to 0.1 ml of the sample or prepared dilutions of standard (100–
500 μg/ml). Upon homogenization for 5 min, it was vortexed with
0.3 ml of 20% Na
2
CO
3
solution. Following that, it was kept away from
light for 60 min at room temperature. Finally, all solutions were spec-
trophotometrically measured (Cary 50-Varian Inc., Walnut Creek, CA,
USA) at 740 nm against a blank. The experiment was done in triplicate
(Singleton, Orthofer, & Lamuela-Ravent
os, 1999). The findings are
represented in terms of milligrams of Gallic acid equivalent per gram
(mg [GAE]/g) of dry matter. While the TFC was evaluated by the addi-
tion of 1 ml of extract solution and/or dilutions of a prepared standard
solution (10–100 μg/ml) to 4 ml of dist. Water. At that time, 5%
NaNO2 (0.3 ml) was applied to the latter mixture. After 5 min, 0.3 ml
of 10% AlCl
3
was added. After 6 min, 2 ml of 1 M NaOH was added.
Finally, dist. H
2
O was added to complete the overall volume of 10 ml,
and it was thoroughly blended. The absorbance of solutions was read
against a freshly made blank at 510 nm (Zhishen, Mengcheng, &
Jianming, 1999). The TFC was denoted by milligrams of quercetin
equivalent per gram of dry matter (mg QE/g).
2.4.2 | Tannins, alkaloids and saponin
The quantitative estimation of tannin, steroid, alkaloids and saponins
was performed according to Swain (1979), Harborne (1973) and
Brunner (1984), respectively.
2.5 |Antioxidant activity of herbal water
extracts (AOXA)
The DPPH (1,1-diphenyl-2-picryl hydrazyl) assay was implemented to
measure AOXA (Sánchez-Moreno, Larrauri, & Saura-Calixto, 1998).
Accordingly, 0.1 ml of each extract at concentration ranged from 0.1
to 0.5 mg/ml and 3.9 ml of DPPH (0.025 g/L methanol) were mixed.
Ascorbic acid was used as positive control (10–100 μg/ml). Then sam-
ples was stirred, and stored in a dark place for 15–20 min. The absor-
bance was read at 515 nm. The subsequent equation was used to
estimate the scavenging ability:
Scavenging activity %ðÞ¼ Absorbance of controlð½
Absorbance of test sampleÞ=Absorbance of control
100
The half-inhibition concentration (IC
50
) value was calculated from
plots of inhibition (%) vs concentration of the each sample.
2.6 |In vivo antishigellosis activity
2.6.1 | Experimental animals
Fifty-four healthy male and female Albino rats (average weight
200 ± 10 g) were obtained from the animal house of the Food
Technology Research Institute, Agriculture Research Center, Giza,
Egypt. Rats were kept in conventional health laboratory settings for
1 week. Temperature was adjusted at 25C ± 2 and 12 h light–dark
and fed a basic diet and water ad libitum before the initiation of the
experiment (Reeves, Nielsen, & Fahey, 1993). All experimental
procedures were followed in compliance with the guidelines of the
Guide for the Care and Use of Laboratory Animals—NRC (2011) (NIH
Publication No. 85-23, Revised 2011).
2.6.2 | Experimental design
For the preparation of bacterial inoculum, S. dysenteriae type I inocu-
lum was matched against 4.0 on the MacFarland scale, which equa-
tes to 12 10
8
CFU/ml when measured spectrophotometrically at
450 nm (Kamgang, Pouokam, Fonkoua, Penlap, & Biwolé, 2005). This
prepared inoculum was utilized in the amount of 1 ml. Whereas,
extract solutions were prepared with distilled water before being
administered orally to rats at dosages of 100 and 200 mg/kg of body
weight (bw).
AMER AND ABD EL-RAHMAN 3of17
For the deparasizatation of the animals, all rats were adminis-
trated a daily dose of 5 mg/kg bw Azithromycin for 3 days at
8 a.m. and 8 p.m. Then, the rats were divided into nine groups,
each comprising of six rats, individually separated in metabolic
cages.
All groups, excluding the negative control group, received 1 ml of
the saline diluted inoculum orally and were observed for diarrhea
induction.
After 3 days, diarrheal stools appeared and the treatments began
with administrating two doses of 100 and 200 mg/kg bw of selected
herbal extracts or with the antibiotic ciprofloxacin (20 mg/kg of
body weight (bw) daily by the oral route for seven consecutive days:
The rats were grouped into:
G1: (Control ve) received 1 ml of distilled water.
G2: (Control +ve) received only 1 ml of bacterial inoculum.
G3 and G4: were treated with garlic extract at doses of 100 and
200 mg/kg bw, respectively.
G5 and G6: rats were given mixtures of garlic and cloves at doses of
100 and 200 mg/kg bw, respectively.
TABLE 1 The Antibacterial activity of
different herbal extracts (100 mg/ml)
against Shigella dysenteriae type I (sd1)
Herbal extracts Inhibition zone (mm) MIC (mg/ml) MBC (mg/ml)
WG 50 ± 0.75b 0.78 1.56
WC 32 ± 0.02d 1.56 1.56
WF 19 ± 0.06f 1.56 3.125
WB 10 ± 0.07h 50 50
WZ 12 ± 0.03g 12.5 12.5
WGC 65 ± 0.02a 0.39 0.78
WGF 42 ± 0.03c 0.78 0.78
WGFC 28 ± 0.05e 1.56 3.125
Cirofloxacin (5 μg) 24 ± 0.02e 0.31
a
1.25
a
Note: Water extract of fenugreek (WF); black seed (WB); clove (WC); garlic (WG); ginger (WZ); mixtures
of garlic and glove (WGC) (1:1); mixtures of garlic and fenugreek (WGF), mixtures of garlic, Clove and
Fenugreek WGCF (1:1:1). Different letters are significantly different (p< .05) among treatments in the
same column. Data for MIC and MBC are the mean of three independent determination but the variation
was less than 1/100,000.
a
μg/ml.
(a) (b) FIGURE 1 Antibacterial activity of
different herbal extracts. (a) Well diffusion
assay: F, fenugreek extract; G, garlic
extract; C, clove extract. (b) Microdilution
assay of different herbal water extract
(WE): A, fenugreek WF; B, black seed
(WB); C, clove (WC); D, garlic (WG); E,
ginger (WZ); F, mixtures of garlic and
glove (WGC) (1:1); G, mixtures of garlic
and fenugreek (WGF); H, mixtures of
garlic, clove and fenugreek WGCF (1:1:1)
TABLE 2 Phytochemical
quantification of fenugreek, garlic and
clove water extract
Extracts WF WG WC
Phenolics (mg GAE/g) 217.79 ± 4.18c 222.79 ± 2.68b 229.73 ± 2.32a
Flavonoids(mg QE/g) 226.51 ± 5.18b 548.80 ± 12.41a 207.88 ± 7.63c
Antioxidant activity (%) 95.65 ± 1.33b 97.91 ± 0.30a 82.35 ± 0.79c
IC
50
(μg/ml) 99.54 ± 2.53c 55.22 ± 1.6a 88.41 ± 1.55b
Saponin (%) 24.57 ± 0.21a 3.3 ± 0.12b 3.5 ± 0.11b
Steroid (%) 20.34 ± 0.12b 0.43 ± 0.05a 1.3 ± 0.08c
Tannins (%) 5.7 ± 0.09a 4.5 ± 0.09c 4.9 ± 0.12b
Alkaloids (%) 1.5 ± 0.23c 6.1 ± 0.13b 8.4 ± 0.15a
Note: Water extract of fenugreek (WF); clove (WC); garlic (WG). Data with different letters are
significantly different among treatments (p< .05). IC
50
, The half-inhibition concentration.
4of17 AMER AND ABD EL-RAHMAN
G7 and G8: received mixtures of garlic and fenugreek with two doses
of 100 and 200 mg/kg bw, respectively.
Finally, G9: rats received the antibiotic ciprofloxacin (20 mg/kg of
body weight (bw).
2.6.3 | Evaluation of stool frequency and weight,
SD1 content and weight gain
The stools of each animal were also collected daily using a white cloth
fixed under the bars supporting the animal in the metabolic cage dur-
ing all the experiment period. Then, the frequency and weight of feces
were recorded. The presence of stools containing mucus or blood was
also noted daily.
Further enumeration of sd1 in feces was accomplished by
weighing 0.5 g of fresh stool from each rat and dissolving it in 4.5 ml
of sterile saline, and then further serial dilution was made, followed by
plating 50 μl of each dilution over salmonella-shigella agar. It was
counted and expressed as CFU/ml after 24 h of incubation at 37C.
After the experiment was completed, the rats were weighed again to
assess how much weight they had gained.
2.6.4 | Studies on serological, hematological and
histology
Rats get weighted after the completion of treatment. Blood samples
were drawn from the animal's ocular plexus under diethyl ether anes-
thesia after the rats had been starved for 24 h. The animals were then
slaughtered, and the colon was quickly torn out, and a portion of it
was preserved in 10% neutral buffered formaldehyde for histopatho-
logic tests. Blood serum was separated by centrifugation at 4000 rpm
for 15 min at 10C and kept at 18C until analysis. The activities of
alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
FIGURE 2 The effect of different
herbal extracts and antibiotic ciprofloxacin
on Shigella dysenteriae type I density in
stool (log
10
CFU/g) expressed as percent
relative to initial number over 7 days of
treatment with herbal extracts and
ciprofloxacin (20 mg/kg bw). Each data
column represents the mean ± S.E.M.
(n=5). Data columns of the same day
with different superscripts (a, b,…) are
significantly different (p< .5) compared
with initial value at first day
FIGURE 3 The effect of different
herbal extracts and antibiotic ciprofloxacin
on total stool frequency before and during
treatment of Shigella dysenteriae type I
diarrheic rats over 7 days of treatment
with herbal extracts and ciprofloxacin
(Ciprof). Each data column represents the
mean ± S.E.M. (n=5). Data columns of
the same group with different
superscripts (a, b,…) are significantly
different compared with the day before
treatment (1, ■). Data column of the
same day with superscript * are
significantly different compared with
diarrheic control G2 (*p< .05; **p< .01)
AMER AND ABD EL-RAHMAN 5of17
were measured using the technique of Bergmeyer and Harder (1986).
Creatinine and urea levels were also estimated using the methods
described by Larsen (1972) and Orsonneau, Massoubre, Cabanes, and
Lustenberger (1992). Hematological parameters like red blood cell
(RBC) count, hemoglobin concentration (Hb), white blood cell (WBC)
count, hematocrit (PCV), and mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin
concentration (MCHC) were determined in whole blood (Armour,
Blood, & Belden, 1994). Nitric oxide levels were assessed according to
Montgomery and Dymock (1962).
2.6.5 | Histopathological analysis
Hematoxylin and eosin (H&E) were utilized for staining sections of
colon (preserved in 10% formalin and paraffin wax) according to Yoon
FIGURE 4 The effect of different herbal extracts and antibiotic ciprofloxacin on the total stool weight (g/day) before and during treatment of
Shigella dysenteriae type I diarrheic rats over 7 days of treatment with herbal extracts and ciprofloxacin (20 mg/kg bw). Each data column
represents the mean ± S.E.M. (n=5). Data columns of the same group with different superscripts (a, b,…) are significantly different compared
with the day before treatment (1, ■). Data columns of the same day with superscript * are significantly different compared with diarrheic
control (*p< .05; **p< .01; ***p< .001)
FIGURE 5 The effect of different herbal extracts and antibiotic ciprofloxacin on weight gain of rats; the weight difference between first and
last day of the experiment. G1: (Control ve) normal uninfected groups; G2: (Control +ve) SD1-infected untreated group; G3 and G4: infected
and treated rats with garlic extract (100 and 200 mg/kg bw, respectively). G5 and G6: infected and treated rats with mixtures of garlic and clove
extracts (1:1, 100 and 200 mg/kg bw, respectively); G7 and G8: infected and treated rats with mixtures of garlic and fenugreek extracts (1:1,
100 and 200 mg/kg bw, respectively); G9: infected and treated rats with the antibiotic ciprofloxacin (20 mg/kg of body weight (bw). Data
columns of the groups with different superscripts (a, b,…) are significantly different compared with G2 (p< .05)
6of17 AMER AND ABD EL-RAHMAN
TABLE 3 Hematological parameters following 7-day treatment of rats with different herbal extracts
Groups Hb (g/dL) RBC (10
6
/mm
3
) PC (10
3
/mm
3
) WBC (10
3
/mm
3
) PCV% MCV (fl) MCH (pg) MCHC (%)
G1 16.57 ± 0.58a 6.80 ± 0.15a 183.0 ± 17.05a 11.63 ± 1.13a 46.63 ± 1.4a 84.33 ± 2.3a 28.66 ± 0.66a 34.33 ± 1.4a
G2 12.47 ± 0.49c 3.62 ± 0.23c 591.3 ± 37.9d 5.90 ± 0.58b 36.36 ± 2.3b 68.00 ± 2.6b 18.00 ± 1.15b 28.66 ± 2.0b
G3 12.97 ± 0.52bc 4.75 ± 0.44bc 311.7 ± 43.5c 13.03 ± 0.66a 43.53 ± 0.52a 84.00 ± 5.1a 27.33 ± 1.4a 32.00 ± 0.57ab
G4 14.63 ± 0.75abc 6.03 ± 0.52ab 305.7 ± 25.7bc 10.03 ± 0.95a 45.23 ± 2.74a 80.33 ± 1.4a 28.66 ± 1.7a 33.33 ± 2.0a
G5 14.47 ± 0.74abc 5.61 ± 0.46ab 244.0 ± 28.8abc 12.73 ± 1.09a 46.23 ± 0.77a 83.33 ± 2.6a 27.66 ± 1.2a 31.66 ± 1.33ab
G6 14.77 ± 0.76abc 6.18 ± 0.63ab 218.3 ± 35.6abc 12.80 ± 1.60a 44.63 ± 1.9a 83.66 ± 2.9a 28.00 ± 1.5a 32.33 ± 0.66ab
G7 14.70 ± 0.91abc 5.97 ± 0.53ab 260.7 ± 26.1abc 12.50 ± 0.73a 44.36 ± 1.9a 84.66 ± 2.9a 28.33 ± 0.88a 34.00 ± 0.57a
G8 14.70 ± 0.64abc 6.96 ± 0.63a 301.7 ± 23.92bc 13.10 ± 0.58a 42.80 ± 1.5a 80.33 ± 2.4a 27.33 ± 0.88a 32.66 ± 1.3ab
G9 15.39 ± 0.99ab 6.64 ± 0.37a 204.3 ± 34.49ab 11.73 ± 0.44a 46.10 ± 1.5a 82.66 ± 2.9a 27.3 ± 1.3a 33.33 ± 0.88a
Note: G1: (Control ve), normal uninfected groups; G2: (Control +ve), SD1 infected-untreated group; G3 and G4: infected and treated rats with garlic extract (100 and 200 mg/kg bw, respectively). G5 and G6:
infected and treated rats with mixtures of garlic and clove extracts (1:1, 100 and 200 mg/kg bw, respectively); G7and G8: infected and treated rats with mixtures of garlic and fenugreek extracts (1:1, 100 and
200 mg/kg bw, respectively); G9: infected and treated rats with the antibiotic ciprofloxacin (20 mg/kg bw). Data columns of the groups with different letters are significantly different (p< .05).
AMER AND ABD EL-RAHMAN 7of17
et al. (2001). The histopathology laboratory of Cairo University's Fac-
ulty of Veterinary Medicine performed the examination.
3|STATISTICAL ANALYSIS
The results were represented as the mean ± SE of three replicates. Bacte-
rial densities were expressed in log10beforeanalysis.TheDatawasinter-
preted following a one-way ANOVA using SPSS version 16.0. Results were
considered statistically significant at p< .05 (Snedecor & Cochran, 1980).
4|RESULTS
4.1 |Antibacterial activity of different herbs water
extracts
The Anti-Shigellosis capability of the produced herbal extracts was
first assessed using the well diffusion method, and then using the mic-
rodilution method to determine MIC and MBC (Table 1). For herbal
water extract alone, WG had the largest inhibitory zone, followed by
WC, and WZ had the lowest. As a result, the herbal combinations
were made from the best herbal extracts, yielding the highest zone.
WGC has the highest inhibitory zone in herbal mixes, followed
by WGF.
In terms of MIC and MBC (Figure 1b), WGC had the lowest MIC
of 0.39 mg/ml, followed by WG and WGF, both of which had
0.78 mg/ml for MIC. On the other hand, WGC and WFC had the low-
est MBC (0.78 mg/ml), followed by WG and WC (1.56 mg/ml).
4.2 |Phytochemical contents and antioxidant
activity
Garlic, clove, and fenugreek water extracts presented the best
antibacterial potential, thus their phytochemical composition and
scavenging activity were evaluated (Table 2). In TPC, the results were
approximately equivalent among the three preparations that ranged
from 217.79 ± 4.18 to 229.73 ± 2.32 GA/100 g, with WC regis-
tering the highest concentrations. Nevertheless, there was a big vari-
ation (p< .5) in TFC, with WG displaying the greatest level
(548.80 ± 12.41 QE/100 gm).Also according to quantitative phyto-
chemical study, fenugreek has the highest concentration of saponin
(24.57%) and steroids (20.34%) and the lowest concentration of
alkaloids (1.5%). Whereas the percentage of tannin was quite com-
parable among the three extracts. Likewise, it was found that clove
extract contains the highest amount of alkaloid that making about
8.4 %.Considering antioxidant activity, all samples demonstrated an
elevated level of scavenging potential (97.91%–82.35 %), with WG
having the highest percentage (97.91%) for extract equivalent to
ab
d
c
bab b
aab a
0
10
20
30
40
50
60
70
80
mg/dL
a
d
c
abc
ab
bab ab ab
0
10
20
30
40
50
60
70
80
mg/dL
a
d
bc babc
c
abc ab abc
0
10
20
30
40
50
60
70
80
mg/dL
a
c
ab ab bbab ab a
0
0.5
1
1.5
2
mg/dL
(a) (b)
(c) (d)
FIGURE 6 The effect of
different herbal extracts and
antibiotic ciprofloxacin on some
liver and kidney functions; a, b, c
and d denoted ALT, AST, urea and
creatinine levels, respectively. G1:
(Control ve) normal uninfected
groups; G2: (Control +ve)
SD1-infected untreated group; G3
and G4: infected and treated rats
with garlic extract (100 and
200 mg/kg bw, respectively).G5
and G6: infected and treated rats
with mixtures of garlic and clove
extracts (1:1, 100 and 200 mg/kg
bw, respectively); G7 and G8:
infected and treated rats with
mixtures of garlic and fenugreek
extracts (1:1, 100 and 200 mg/kg
bw, respectively); G9: infected and
treated rats with the antibiotic
ciprofloxacin (20 mg/kg bw). Data
columns of the groups with
different superscripts (a, b,…) for
the same parameter are
significantly different compared
with G2 (p< .05)
8of17 AMER AND ABD EL-RAHMAN
200 μg/ml. The lowest IC
50
was 55 μg/ml which was for garlic
followed by 85 and 99.5 μg/ml or fenugreek and clove water extract,
respectively. While IC
50
value for ascorbic acid (positive control) was
52 μg/ml.
4.3 |Effect of different herbal water extracts on
Rats' Shigella sd1 load
The burden of S. dysenteriae type I in infected rats without treatment
(G2) increased considerably by percent of 137% (p< .05) from the
first day (log [CFU] ’9.08) to the seventh day (log [CFU] ’12.51).
This percentage was significantly reduced in the treated groups. G7
(22.9%), G9 (23.9%), and G8 (27.6%) had the lowest percent of Shi-
gella load at the end of the experiment compared to the first day,
corresponding to log (CFU) of 2.08, 2.18, and 2.51, respectively
(Figure 2).
As shown in Figure 3, total stool frequency (TSF) increased sub-
stantially (p< .05) throughout the experiment period in sd1-infected
animals (G2). TSF was lower in herbal-treated groups as compared to
G2, with its most significant outcomes seen in groups 6, 7, and 8 on
the first, third, and fifth days. The highest percent of reduction (90%)
was achieved by G7 in day 7. The TSF of rats given ciprofloxacin
(20 mg/kg BW) was virtually equal to the day before the trial,
although it showed an increasing tendency when contrasted to rats
given herbal treatment. As a result, G7 and G8 had the lowest TSF of
all groups during the experiment days.
Before the induction of diarrhea, the mean weight of the stool
was 3.46 g/day. The day after, Shigella infection caused a significant
rise in stool weight in rats (Figure 4). During the 7 days of infection,
the weight of the stool sharply increased by 267% (12.7 ± 0.67 g/
day) in G2 as compared to the day before the experiment. Contrary
to the G2, herbal-treated groups demonstrated a significant
decrease (p< .05) in the weight of the stool, giving a decreasing per-
cent ranging from 26% to 68% on the seventh day of the experi-
ment. In groups G7 and G8, It was also noticed on the first day that
there was a significant decrease in percent of stool weight. On the
first day, the stool weight increased in (G9) as compared to the day
before diarrhea-induction, but it substantially decreased through the
days of the experiment as compared to the diarrhea group. At the
end of the experiment, G7, G9 and G8 demonstrated the lowest
stool weights (1.09 ± 0.08, 1.107 ± 0.15, and 1.09 ± 0.15 g/day,
respectively).
4.4 |Effects of the different herbal water extracts
on the body weight
The initial body weights of all groups were compared to their final
weights and are presented in Figure 5. During the S. dysenteriae type
1 administration phase, no animals perished. S. dysenteriae type
1 (G2) administration resulted in a substantial drop in rat body weight
as compared to the negative uninfected rats (G1). A progressive
weight gain was observed in rats treated with various herbs in com-
parison to G2, with G7 and G9 significantly (p< .05) regained weight
and being comparable to G1.
4.5 |The effect of the different herbal water
extracts and ciprofloxacin on some biochemical
parameters
As can be recognized from Table 3, Sd1 infection significantly altered
blood parameters, resulting in a substantial drop in Hb, RBC, WBC,
PCV, MCV, and MCHC, with a substantial increase in PC when com-
pared to the uninfected group (G1). There was a remarkable
FIGURE 7 The effect of different herbal extracts and antibiotic ciprofloxacin on nitric oxide (NO) level in colon and serum in diarrheic rats
after 7 days. G1: (Control ve) normal uninfected groups; G2: (Control +ve) SD1 infected untreated group; G3 and G4: infected and treated rats
with garlic extract (100 and 200 mg/kg bw, respectively).G5 and G6: infected and treated rats with mixtures of garlic and clove extracts (1:1, 100
and 200 mg/kg bw, respectively); G7 and G8: infected and treated rats with mixtures of garlic and fenugreek extracts (1:1, 100 and 200 mg/kg
bw, respectively); G9: infected and treated rats with the antibiotic ciprofloxacin (20 mg/kg bw). Data columns of the groups with different
superscripts (a, b, …) for the same parameter are significantly different compared with G2 (p< .05)
AMER AND ABD EL-RAHMAN 9of17
improvement in treated groups when compared to G2. However,
there was actually no notable variation among treatment groups G4,
5, 6, 7, and 8, which exhibited a considerable improvement in Hb and
RBC. In view of other parameters, including PCV, MCV, MCH, and
MCHC, there was no big difference between all other treated groups
(p> .05). The outcomes of the treated groups were comparable to
those of the uninfected groups (G1). However, in PC, the lowest
values that corresponded to the normal group were G6, G5, and G7 in
that order. The most comparable groups to G1 were G9 in Hb, G8 and
G9 in RBC, G9 and G6 in PC, G5 and G9 in PCV, G7 and G3 in MCV,
G4 and G7 in MCH and finally G7 in MCHC.The effects of several
water herbal extracts on some liver and kidney functions are summa-
rized in Figure 6. The data obtained revealed a significantly distinct
(p< .05) difference in the levels of ALT, AST, and urea between the
negative group and the other groups. Untreated Shigella-infected rats
had the highest level of all parameters when compared to both the
FIGURE 8 The effect of different herbal extracts and antibiotic ciprofloxacin on histological changes in the colon of rats. (a) Colon
section from group 1 (G1), showing normal histological layers (mucosa, submucosa, and serosa); (b) colon section from G2, arrow showing massive
inflammatory cells infiltration in the gastric mucosa and submucosal layers; (c) colon section from G3 showing congestion of submucosal blood
vessels; (d) colon section from G4 showing slight activation of mucous secreting glands; (e) colon section from G5 showing slight activation of
mucous secreting glands; (f) colon of rats from G6 showing few inflammatory cells infiltrations in the gastric mucosa and submucosal layers; (g)
colon section from G7 showing no histopathological changes; (h) colon section from G8 showing slight activation of mucous secreting glands;
(i) colon section from G9 showing few inflammatory cells infiltrations in the gastric mucosa; (H&E 100)
10 of 17 AMER AND ABD EL-RAHMAN
uninfected control (G1) and other treated groups. In terms of ALT
(Figure 6a), it measured 60 U/L, which corresponds to a 249%
increase when compared to the G1. All of the treated groups showed
a significantly lower percentage of the rise. G7 and G9 had the lowest
rising percent in treatment groups, which was around 11% for both
groups in ALT. Regarding AST levels (Figure 6b), G2 (+ve control)
increased by 274% as compared to G1, with G9 (18%), G7 (26%), and
G5 (26%) having the lowest proportions. Similarly, the G2 showed a
percentage rise comparable to 98% in urea (Figure 6c), while the G8
showed the lowest percentage gain (7.6%). In the case of creatinine
readings (Figure 6d), G2 accounted for 99% of the rise, whereas G7
had the lowest percentage increase (24%), followed by G9 (26%).
As indicated from Figure 7 infecting rats with sd1 resulted in a
great increase in NO concentration in the colon, with 513% of
increase equivalent to 580.66 ± 6.06 μM for G2 as opposed to
94.7 ± 4.8 μM for G1. Other groups had a decreased percent of
increase ranging from 135.7 to 226.7%. The least percent was
recorded by G5 (135.66 ± 6.6 μM) followed by G9 (141.0 ± 9.6 μM).
Regarding NO concentration in serum, the percent of the increase
was 178.43% (175.33 ± 5.04 μM) for G2, as compared to the normal
group (G1, 61.0 ± 3.7 μM). Other treated groups showed a lower per-
centage of rises, ranging from 19.6 to 73.13%. In which, G9 displayed
the lowest percent (73.00 ± 5.29 μM) followed by G6
(75.0 ± 4.04 μM) and G7 (76.00 ± 3.78 μM).
4.6 |Effect of the different herbal water extracts
and ciprofloxacin on the colon histopathology
The infected group G2 (Figure 8b) exhibited substantial histological
alterations compared to the normal group G1 (Figure 8a). G1 demon-
strated the typical histological view. While rats from G2 had extensive
infiltration of inflammatory cells in the gastric mucosa and submucosal
layers as well as submucosal edema that included congestion of submu-
cosal blood vessels. However, there was a noticeable improvement in
the treated groups. Scarce inflammatory cells infiltrated the gastric
mucosa, besides submucosal blood vessels were congested in
G3 sections (Figure 8c). Additionally, a small number of inflammatory
cells with slight activation of mucous-secreting glands were found in
the gastric mucosa of G4 (Figure 8d) who had minor alterations in the
colon. Anatomical alterations in the colon of G5, 6, 7, 8, and 9 were not
found (Figure 8e–h, respectively). There was just a minor activation of
mucus-secreting glands in some regions of G5. Also, very few inflam-
matory cells were noted in the gastric mucosal and submucosal layers
of G6. G8 section likewise, had minor activation of mucous-secreting
glands. Group 9 samples also exhibited a scarce inflammatory cell.
5|DISCUSSION
Shigella strains are classified as multidrug-resistant (MDR). Resistance
levels ranging from 80% to 100% have been observed in Nigeria
(Abdu, Aboderin, Elusiyan, Kolawole, & Lamikanra, 2013), Egypt
(Ahmed et al., 2013; El-Gendy et al., 2012), Iran (Nikfar, Shamsizadeh,
Darbor, Khaghani, & Moghaddam, 2017), Central Africa Republic
(Breurec et al., 2018), and Somalia (Ali Nor et al., 2021). Additionally,
Zaki et al. (1986) studied Egyptian newborns and found that Shigella
incidence was greater in Egyptian infants aged less than 6 months
(30/1000 child-years) than in infants aged more than 6 months
(23 episodes/1000 child-years).
The anti-shigellosis potential of certain herbal water extracts was
investigated in this study as a natural substitute for antibiotics. Five
different aqueous herbal extracts were tested against Shigella sd1,
including garlic, clove, fenugreek, ginger, and black seed, as well as
three distinct herbal mixes including garlic and clove (WGC), garlic and
fenugreek (WGF), and garlic, fenugreek and cloves (WGCF). Consider-
ing the MBC/MIC ratio, all extracts had a value of 4, indicating that
they have a bactericidal effect (Levison, 2004). Water extracts of
WGC, WG, and WGF were deemed the best antibacterial extracts
based on the highest inhibition zone and lowest MIC.
The variation in antibacterial outcomes between the two
approaches of the antibacterial assay is due to a difference in the rate
of diffusion of each extract in agar. Fenugreek extract, for example,
has a higher viscosity, which hinders its dispersion. Garlic's activity
has been attributed to the presence of Allicin and other thiosulphates,
which can react with the thiol group of certain bacterial enzymes
(Ankri & Mirelman, 1999). Garlic has been shown in recent studies to
have bacteriostatic properties against Shigella sonnei and Shigella
flexneri (Olaimat et al., 2017).
Whereas the antibacterial property of clove ethanolic extract has
been observed against several types of bacteria, and the MIC for
Diethyl Ether Extract has been recorded as 0.05 and 500 μg/ml (Abou
El Nour, 2018). The acquired results were also regarded as confirma-
tory to the primary investigation conducted by Rahman, Rahman,
Islam, Alam, and Korea (2011), however the current results out-
performed the previously reported MIC (180 mg/ml) and MBC
(680 mg/ml) of clove extracts against Shigella ds1 isolates. Clove
extract functions by possessing potentially bioactive components as
eugenol (2-methoxy-4-[2-propenyl] phenol), glycosides, flavonoids,
saponins, tannins, and essential oils (Abou El Nour, 2018).
Several studies have shown that Fenugreek has in-vitro
antibacterial potential against Salmonella typhi,Escherichia coli,Pseudo-
monas aeruginosa, Staphylococcus aureus,Klebsiella pneumonia, and Shi-
gella sonnei. This activity is attributed to the plant's high concentration
of phenolic and flavonoid compounds (Chalghoumi, Mabrouki,
Abdouli, & Line, 2020). On the other hand, the MIC of Nigella sativa
seed volatile oil for Shigella,Vibrio, and Escherichia strains has been
reported to be between 50 and 400 μg/ml (Ferdous, Islam, Ahsan,
Hasan, & Ahmed, 1992). Similarly, ginger has been shown to be an
efficient antimicrobial agent against a variety of pathogenic bacteria,
comprising Shigella spp. giving an inhibition zone of 12 mm
(Yadufashije, Niyonkuru, Munyeshyaka, Madjidi, & Mucumbitsi, 2020).
The combination of extract of garlic with either fenugreek or clove
demonstrated the greatest antibacterial potential, as reported for the
first time in this research. This could be considered as a synergistic
effect of these extracts. Whereas our results for the antibacterial
AMER AND ABD EL-RAHMAN 11 of 17
potential of Ciprofloxacin were substantiating to other investigations,
this antibiotic belongs to the family fluoroquinolone and is known to
prevent the supercoiling of bacteria chromosomes by hindering the
activity of DNA gyrase (Kouitcheu, Tamesse, & Kouam, 2013).
Garlic's antioxidant potential reported across this research was
confirmatory by Johnson et al. (2016) who found it to range from
4.47% to 92.44%. The current TPC and TFC of fresh garlic extract
support the findings of Alide, Wangila, and Kiprop (2020), who
reported a mean TPC and TFC of 4.47–92.44 mg GAE/100 g and
109.78 ± 6.78 mg QE/100 g, respectively. In the case of the clove
water extract, the TPC and TFC obtained were equivalent to those
found by Al Mashkor (2015). Though limited work has been con-
ducted on fenugreek water extract, a recent study found that the total
polyphenols of fenugreek water extract, range between 31.7 and
18.9 mg GAE/100 g extract, with the identification of Kaempferol,
Genistein, Vanillin, Myrécitine and Rutin, which are thought to be the
causes of its remarkable antioxidant activity (Benziane, Acem,
Aggad, & Abdali, 2019). Also saponin in fenugreek is considered a
potent antioxidant agent that have various biological activity (Francis,
Kerem, Makkar, & Becker, 2002). The percentage of other phyto-
chemicals like saponin, alkaloids, steroids and tannins in fenugreek,
garlic and clove extracts are in accordance with other authors (Ali,
Nas, Yahaya, & Ibrahim, 2018; Mahmood & Yahya, 2017; Yusuf,
Fagbuaro, & Fajemilehin, 2018).
DPPH free radical scavenging assay is used to evaluate the anti-
oxidant capability, this assay is commonly used for extracts produced
from the medicinal plants in which higher inhibition level demonstrate
a strong antioxidant (Nurliyana, Kamarudin, Idid, Rehan, &
Koya, 2010). All three extracts used in this investigation, garlic, clove,
and fenugreek, had substantial antioxidant activity, which is consistent
with previous authors' findings (Dua, Singh, & Mahajan, 2015; Jang
et al., 2017; Norziah, Fezea, Bhat, & Ahmad, 2015). This antioxidant
activity is often ascribed to the presence of phytochemicals, which
give different therapeutic advantages in a variety of diseases such as
arthritis, cardiovascular disease, edema, nausea, asthma, bacterial
infections, and cancer (Kasote et al., 2015).
The WG, WGF, and WGC herbal extracts were utilized to treat
Shigella-infected rats in order to improve their efficacy in managing
shigellosis. The dose of extracts (100–200 mg/kg) was chosen based
on other studies (Rana, Pal, Vaiphei, & Singh, 2006, Nair and Jacob,
2016, Mohammad-Sadeghipour et al., 2020). The stool of infected
rats appeared elongated, dark, and shiny due to the presence of blood
and mucous (Kamgang et al., 2005). Infected rats also suffered from
weight loss, increased frequency and weight of feces, and an increase
in the number of Shigella over time, and these findings were corrobo-
rated by other researchers (Kamgang et al., 2005).The symptoms of
shigellosis in rats were dramatically reduced in rats treated with either
herbal extracts or antibiotics. G7 produced the largest percent reduc-
tion, with a fall in sd1 density in feces (77.1%), as well as a decrease in
stool frequency (90%) and weight (66%). Kouitcheu et al. (2013)
obtained comparable findings with Picralima nitida (500 mg/kg),
reporting a percent reduction of 65.6, 61, and 49.35% for Sd1 density,
stool frequency, and feces weight, respectively. Herein, the best per-
formance was for the fenugreek and garlic mixture extract (WGF),
suggesting both in vitro and in vivo antibacterial activity and anti-
diarrheal effects that fenugreek and garlic are characterized by. This
antidiarrheal activity is linked to the presence and quantity of alka-
loids, saponins, flavonoids, sterols and/or terpenes in both extracts of
fenugreek (Boyina, Kosanam, & Rani, 2014 ) and garlic (Azubuike &
Ajaegbu, 2019). The reported anti-diarrheal action could be attributed
to different aspects, among them is the inhibition of prostaglandin
production (PGE2). As a result of sd1 infection or other stimuli such as
castor oil, the intestinal mucosa becomes inflamed, which releases
prostaglandin biosynthesis and subsequently stimulates motility and
discharges (Boyina et al., 2014). Furthermore, it can be related to the
inhibition of glucose absorption, which is a major stimulus for intesti-
nal absorption of water and electrolytes. Additionally, tannins can
denaturize proteins, forming protein tannates, which increase the
intestinal mucosa's resistance to chemical change and reduce secre-
tion. Besides, flavonoids' ability to reduce intestinal motility and influ-
ence hydro-electrolytic secretion, they are also effective in treating
diarrhea (Das, Prakash, & Devaraj, 2003). Moreover, the zinc content
in garlic, which is estimated to be 0.34 mg/100 gm (Sajid, Butt,
Shehzad, & Tanweer, 2014), is thought to aid the retardation of diar-
rhea. Since zinc supplementation (20 mg/day) has been suggested as
an adjunctive treatment for diarrhea in kids (Bhutta et al., 2000).It is
substantial to note that raising the dose of the garlic extract from
100 mg to 200 mg/kg bw did not result in a marked decrease in diar-
rhea. Despite the antidiarrheal activity of garlic, the toxic effect of
large dosages of this herb has already been observed in a few studies.
This is due to the parasympathetic stimulation of the gastrointestinal
tract generated by allicin, which leads to increased GI contractility and
movements and, as a result, an increase in diarrhea and hyper-defeca-
tion (Alare and Alare, 2020). From here came the importance of the
herbal combination that will allow the use of garlic at a lower dose
with increased activity. Antibiotics, on the other hand, cause aggres-
sive killing of bacteria, which may lead to endotoxic shock and a rise
in the frequency of defecation (Kamgang et al., 2006). As can be
noticed from the current results, Garlic and cloves combination was
ranked second (WGC) in preventing diarrhea. Clove's in vivo anti-
diarrheal effect was found to reduce the onset and duration of diar-
rhea in one investigation (Akhtar, El-Debani, & Senussi, 2011).
Shigella-infected rats had extensive body weight loss, which can
be reversed with the proposed extracts and antibiotic. This weight
gain is driven by a number of factors, including extract nutritional
ingredients, antibacterial action, and other herb properties. Fenugreek,
for example, may positively impact weight growth (Rouag
et al., 2021), because it contains a high proportion (40%) of soluble
fiber, which creates a gelatinous structure that may protect the colon
and abdomen from Shigella infection. They have also been shown to
cause a delay in stomach emptying and carbohydrate digestion
(Hannan et al., 2007).
A blood picture, in addition to stool and weight markers, is con-
sidered an indication of infection. Total leukocyte and neutrophil
counts are expected to rise in infected people. Loss of blood and the
decrease in hemoglobin content is owing to cell lysis caused by Shi-
gella and it also accompanied by an iron uptake locus in the Shigella
genome structure (da Cruz Gouveia, Lins, & da Silva, 2020;
12 of 17 AMER AND ABD EL-RAHMAN
Torres, 2004). Accordingly, infected rats in the current investigation
had the lowest blood parameters, including an elevated platelet count,
which is associated with shigellosis (Adams, Vose, Edmond, &
Lyckholm, 2017). In the other direction, other groups, particularly
those from G4 to G9 tend to improve blood parameters. It was
noticed that, garlic alone at lower dosages (100 mg/kg) in group 3 did
not significantly improve the other blood indices. However, higher
doses (200 mg/ml) in G4 exhibited a significant effect in enhancing
the blood parameters, similar to the results obtained by
Iranloye (2002). Garlic appears to boost white blood cell counts, which
were already low owing to blood loss. Additionally, the observed
increase in WBC indicates an increase in the activation of natural killer
cells T-lymphocytes, which will lead to an improvement in immunolog-
ical function (Iranloye, 2002). Nonetheless, the addition of the clove in
WGC did not result in a further increase in WBC, which is consistent
with the findings of Adam, Mohamed, and Abdelgadir (2013), who dis-
covered that clove in a lower dose decreased WBC. According to
Adebayo, Abolaji, Opata, and Adegbenro (2010), clove extract may
contain bioactive chemicals that inhibit or interfere with WBC forma-
tion. Conversely, groups that were administrated WGF have regained
normal levels of blood indicators, including an increase in Hb, RBC,
and WBC with decreasing PC. The iron content (33 mg/100 g) of
fenugreek extract may compensate for the losses in Hb level. Besides,
its antioxidant properties decrease the oxidative stress in RBC,
preventing the rapture of their cell wall. These recorded hematological
improvements for fenugreek were equivalent to other investigations
(Nagamma et al., 2019).
The liver and kidney are two important organs, which play key
roles in the metabolic pathways of the body. Liver is the site of drug
metabolism while the kidney ensures drug reabsorption and excretion.
ALT and AST are two transaminases and considered as biomarkers for
liver activity. When found at a high level it would be a sign of possible
toxicity (Mukinda & Syce, 2007). Data concerning the activities of
serum AST, and ALT revealed an elevation in Shigella-infected and
untreated rats. That increase underlines hepatic cell lysis, which is an
indication of the likelihood of the toxicity of this organ (Afagnigni
et al., 2017). In the same way, the rise in urea and creatinine levels is
an important marker for kidney dysfunction (Reena, Sanjiv, &
Bhupinder, 2012). In the current study, the treatment of rats with
selected extracts of garlic alone or with its combinations with fenu-
greek or clove caused a decrease in these parameters levels. Especially
those of groups G5, G7, G8 and G9 that were almost significantly
restored to normal values (G1), as compared to positive control rats
(G2). The protective activity against hepatotoxin and nephrotoxin
such as CCL4 and NaNo
2
have been reported for garlic (Almatroodi
et al., 2020), fenugreek (Ati
la Uslu, Uslu, & Adali, 2019) and clove
extracts (El-Hadary & Ramadan Hassanien, 2016). The consequences
of this study likewise indicated that the aqueous extract of garlic,
fenugreek, and clove contains a potent restorative and protective
influence against the biochemical defects caused by the Shigella infec-
tion on the liver and kidney of the rats.
Through the pathogeneses of Shigella and particularly the produc-
tion of Shiga-toxin, it causes several inflammatory responses among
them is triggering of nitric oxide production (Biswas, 2000). No is a
pro-inflammatory mediator that regulates many physiopathological
responses in the human body among them is promoting the migration
of leukocytes to the inflammatory regions (Korhonen, Lahti,
Kankaanranta, & Moilanen, 2005). The high level of NO in serum and
colon is considered an indicator for the high concentration of Shiga-
toxins and consequently Shigella organisms in the group of infected
rats (G2). In herbal and antibiotic-treated groups, the NO level was
greatly reduced, which resulted from the destruction of bacterial
organisms by these antibacterial candidates. Besides, garlic has been
reported to have anti-inflammatory activities through different mech-
anisms among them is the inhibition of the enzymatic action inducible
NO synthase (iNOS), as a result, stopped the NO production (You,
Yoo, Baek, & Kim, 2019). Likewise, the anti-inflammatory of fenu-
greeks and cloves is well documented through their antioxidant activ-
ity that aid in the maintenance of cell stability and integrity (Batiha
et al., 2020; Pundarikakshudu, Shah, Panchal, & Bhavsar, 2016). Also,
Flavonoids and tannins have been confirmed to effectively obstruct
prostaglandins, a group of powerful inflammatory substances
(Manthey, 2000). Similar observation was recorded with treatment of
S. flexneri with hydroethanolic Cola anomala extract (Wambe
et al., 2019).
As a tragic result of the Shigella pathogenesis cascade, severe tis-
sue damage is observed in Shigella-infected rats' colon sections; how-
ever, the tissues began to heal in groups treated with either mixture
of garlic extract with the clove or with the fenugreek extract, as well
as in antibiotic-treated groups, with a remarkable reviving of cell
integrity in G7 (garlic and fenugreek extracts mixture at 100 mg/kg
bw) that was comparable to normal uninfected groups. Fenugreek
extract was reported to be protective against acetic acid-induced coli-
tis in rats in a recent study (Elsayed & Hussein, 2019). This beneficial
effect was connected to the presence of saponin diosgenin, which
properly regulates inflammatory markers and inhibits gut flora. Aside
from its antioxidant and terpene content, which protect against DNA
oxidative damage (Elsayed & Hussein, 2019; Xue, Lei, Li, &
Zhang, 2011).
The evidence from this study suggests that the mixture of fenu-
greek and garlic extract has a superior influence on the treatment of
shigellosis. It is worth mentioning that the usage of this herbal blend
has been established in past studies to have a good impact as a cardi-
oprotective in rats (Mukthamba & Srinivasan, 2015). This herbal blend
can be used in the form of a medicinal preparation, such as a tablet,
for practical purposes. This herbal combination can also be utilized in
food, since it is already employed as a flavoring and preservation ele-
ment in traditional pastirma meat in Egypt and Turkey (Kilic, 2009),
and its peculiar flavor is highly valued.
6|CONCLUSION
The overall conclusion of this study proved that water extracts of gar-
lic, clove, and fenugreek exerted a bactericidal effect on Shigella sd1.
It also demonstrated, to varying degrees, the ability of herbs to delay
AMER AND ABD EL-RAHMAN 13 of 17
shigellosis in infected animals, as evidenced by decreased stool fre-
quency and weight, positive weight gain, recovery of hematological
indices, improvement of liver and kidney parameters, reduction of NO
levels, and restoration of histopathological alterations in colon sec-
tions. The garlic and fenugreek herbal combination, which was to the
best of our knowledge firstly reported, provided the best performance
in nearly all metrics in combating shigellosis across groups and was
almost equal to the uninfected and antibiotic-treated groups. As a
consequence, this effective combination is proposed as a therapeutic
alternative for antibiotics that would help in facing the possible emer-
gence of bacterial resistance to antibiotics. At present, our team is
concerned with the identification of phytochemical profiles of these
extracts to estimate the major compounds and their mode of action
against Shigella.
ACKNOWLEDGMENTS
No acknowledgments to declare.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest.
ETHICAL APPROVAL
This experiment was carried out in the animal house of the Food
Technology Research institute, Agriculture Research Center, Giza,
Egypt. An animal housing and experiments were conducted in strict
accordance with the Guide for the Care and Use of Laboratory Ani-
mals 8th Edition 2011.
AUTHORS' CONTRIBUTIONS
SAA and HSMA have designed the study. SAA and HSMA carried out
the experiment. SAA and HNA interpreted results. SAA drafted the
manuscript. SAA and HSMA reviewed the manuscript.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
ORCID
Sara Adel Amer https://orcid.org/0000-0003-4387-7155
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How to cite this article: Amer, S. A., & Abd El-Rahman, H. S.
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