Rendiconti Lincei.

Scienze Fisiche e Naturali

https://doi.org/10.1007/s12210-023-01135-7

RESEARCH PAPER

Development of more potent anti‑microbial drugs from extracts of five

medicinal plants resistant to S. aureus in human fluids: an ex vivo
and in vivo analysis
Rasha Assiri1 · Nada Abdullah Alharbi1,13 · Thamir Saad Alsaeed2 · Waleed Al Abdulmonem2 ·
Almonther Abdullah Hershan3 · Rana Abdullah Alghamdi4 · Abdullah S. M. Aljohani5 · Nada Alkhorayef6 ·
Ahmad Almatroudi7 · Khaled S. Allemailem7 · Samia S. Alkhalil8 · Ameena A. AL‑surhanee9 · Mariam S. Al‑Ghamdi10 ·
Basal Sulaiman M. Alkhudhairy11 · Hailah M. Almohaimeed12

Received: 5 October 2022 / Accepted: 16 January 2023

© The Author(s), under exclusive licence to Accademia Nazionale dei Lincei 2023

Abstract
Methicillin-resistant staphylococcal infections are a serious public health concern worldwide, especially in our region, due to
the unavailability of accurate therapeutics. The aim of this study was to determine the resistance pattern of Staphylococcus
aureus isolated from clinical and asymptomatic specimens and to identify antimicrobial agents against methicillin resistant
S. aureus strains. 47 samples (pus, urine, blood, nose and milk) together from diverse healthcare hospitals in Faisalabad
for location of S. strains. Aureus by biochemical examination. The results of these biochemical tests showed that 21 of 47
samples were positive for S. aureus. A resistance study found that 10 of 21 S. aureus isolates were resistant to oxacillin.
Twenty-five medicinal plants were collected from different parts of Saudi Arabia, air-dried and extracted with methanol.
These plant extracts were then screened for potential against oxacillin-resistant S. aureus. In addition, the semi-maximum
inhibitory concentration (IC50) of effective medicinal plant extracts was measured using the micro-dilution method. Metha-
nol extracts from five plants (Centratherum antherminticum, Eucalyptus globulus, Lawsonia inermis, Punica granatum and
Rubia cordifolia) have been shown to have anti-bacterial activity against clinically isolated S. oxacillin resistant strains. It
was done. The IC50 for these methanol extracts of S. aureus ranged from 0.250 to 4.30 mg/mL. Our results suggest that
various medicinal plants contain potential anti-bacterial agents against S. aureus, which may contribute to the development
of more effective agents against multidrug resistant S. aureus infections. It might help.

Keywords S. aureus · MRSA · Medicinal plants

1 Introduction equally for both healthcare and community associated

infections globally (Tong et al. 2015). S. aureus are and are
Staphylococcal infections are communicable diseases which gram positive bacteria being cluster forming cocci. They
are the leading cause of primary infections originating in are non-spore forming, non-motile facultative anaerobes,
hospitals, especially in developing countries such as Saudi non-flagellates and are catalase positive organisms (positive
Arabia. Staphylococcal infections are usually referred to as for catalase only?) (Jayasundara 2014). S. aureus is mostly
those caused by the organism Staphylococcus aureus which found in the moist squamous epithelium in the anterior nares
is the one of the most frequently isolated pathogens from (Taylor and Unakal 2017). According to estimation about
clinical samples in Saudi Arabia especially the skin lesions, 20% people have S. aureus in their noses and other 60% are
wound infections and urinary tract infections (UTIs). S. considered as intermittent carriers of S. aureus. It is a most
aureus is the major human infectious agent responsible opportunist pathogen which can cause more severe infec-
tions. These suppurative infections include a wide range
such as furuncle, carbuncle, chronic furunculosis, boils,
* Nada Abdullah Alharbi abscesses, impetigo, pneumonia, deep lesions, mastitis, cel-
[email protected] lulites, endocarditis, osteomyelitis, scalded skin syndrome,
Extended author information available on the last page of the article toxic shock syndrome, bacteria and food poisoning (Miller

13
Vol.:(0123456789)
 Rendiconti Lincei. Scienze Fisiche e Naturali

and Diep 2008). Staphylococcus aureus are present in skin methicillin. Initially used, however, methicillin’s have been
and in mucus, with human beings being the main source largely replaced by similar more stable penicillin’s such as
for these bacteria, including drug resistant strains such as oxacillin, flucloxacillin and dicloxacillin as a result of their
MRSA (Boucher and Corey 2008) (see Table 1). toxicity, and now they're no longer marketed for human use
Resistance to methicillin S. aureus (MRSA) was first (Lee et al. 2018). The risk of infection rises with coloniza-
identified in England in 1961 shortly after the advent of tion of MRSA. In 50–80 per cent of the cases the infections

Table 1  Plants selected for anti-bacterial activity against multidrug resistant S. aureus
Specie Vernacular names Part used Local uses

Acacia nilotica Kikar Leaves, Beans Used to relieve the symptoms of wounds, ulcers, dysentery, hemorrhage,
diarrhea, burning sensation, skin diseases and dental caries
Acacia suma Korh Roots Used as anti-inflammatory and shows analgesic activity
Aloe vera Aloe vera Leaves Used for the treatment of wounds, cuts, burns, eczema, reducing inflamma-
tion and digestive problems
Artemisia absinthium Afsanteen Leaves Used to treat epilepsy, migraine, nervous irritability, depression, liver and
spleen inflammation, facial paralysis and piles
Berberis lycium Sumblo Root bark Plant pacifies anti-diabetic, jaundice, antibiotic, acne, sore throat, diuretic,
liver disorders, mouth swelling, cough and pain killer
Capsicum frutescens Red chilli Fruit Used as an analgesic. Applied to the skin for ache caused by shingles. Used
to lessen muscle spasms
Centratherum anthelminticum Kali jeeri Seeds Used as anthelmintic, spermicidal, hypotensive and laxative
Citrullus colocynthis Tumma Fruit Used as anti-diabetic, anti-epileptic, anti-blenorrhoeic, anthelmintic, aborti-
facient
Eucalyptus globulus Eucaluptus Leaves, Seeds Used as anti-bacterial, anti-viral, anti-fungal, anti-inflammatory, antiseptic
and expectorant. Also used to relieve respiratory infections, acne, bronchi-
tis, rheumatism, muscle aches and pains
Fumaria officinalis Shahtra Whole plant Used to get rid of skin blemishes. Used to treat skin diseases, and conjunc-
tivitis
Lawsonia inermis Mehndi Leaves Used as astringent, diuretic, anti-inflammatory. Also used for the treatment
of bronchitis, burning sensation, rheumatalgia, inflammations, dysentery,
scabies, boils, fever, falling of hair, grayness of hair, jaundice
Moringa oleifera Sahajna Leaves Anti-Inflammatory, Cancer treatment, Alzheimer Prevention
Nigella sativa Kalonji Seeds Used for the treatment of asthma and coughing, to treat tumors and abscesses
of the abdomen, liver, and eyes
Peganum harmala Hermal Leaves Used to treat skin inflammation. Also used as an anthelmintic and abortifa-
cient agent
Piper cubeba Sheetal chini Seeds Used as stimulant, expectorant, stomachic, and carminative. Used in the
treatment of piles, abscess and chronic bronchial inflammation
Piper nigrum Black pepper Seeds It possesses anti-tumorigenic, immunostimulatory, stomachic, carminative,
anticholesterolemic and again known for its strong phytochemical activities
Psidium guajava Gauva Leaves Used against cancer, bacterial infections, inflammation and pain
Punica granatum Anaar Anaar coat It has immuno-stimulatory, anti-oxidant, anti-inflammatory anti-diabetic and
anticancer activities
Rubia cordifolia Majith Heartwood It possesses astringent, antidysenteric, anthelmintic, ophthalmic, and revital-
ized effects
Santalum album Sandal Wood Valuable in skin ailments
Solanum nigrum Mako Leaves Used as anti-tumorigenic, anti-oxidant, hepatoprotective, diuretic, and
antipyretic
Sphaeranthus indicus Mundi booti Leaves Used to treat epilepsy, hepatopathy, mental illness, hemicrania, jaundice,
diabetes, leprosy, pectoralgia, cough, hernia, helminthiasis, dyspepsia and
skin illness
Swertia chirata Charaita Roots Used for the treatment of diabetes, typhoid, pneumonia and malaria
Syzygium cumini Jamun Leaves Plant pacifies diarrhea, diabetes, leucorrhoea, fever, skin diseases and gen-
eral debility
Withania coagulans Paneer dodi Fruit/Flower Used as anti-diabetic and antihyperlipidemic

13
Rendiconti Lincei. Scienze Fisiche e Naturali

fit the colonization strains. Almost any object in skin touch, 15 pus samples were collected from different areas of Fais-
from white clothing to ties with plumbs and cell phones, can alabad (Millat lab, GCUF, Allied hospital and University of
behave like the fomite in MRSA communication. For long Faisalabad). Samples were stored at – 20 °C.
periods of time, colonization may continue. In the home
environment, MRSA may also persist, complicating eradi- 2.2 Biochemical characterization of S. aureus
cation efforts. At the same time, colonization is not static,
because strains evolved within the same host, and were even For the isolation and identification of S. aureus, different
replaced (Turner et al. 2019). Over the past 4 decades, resist- biochemical tests were performed. These tests help to dis-
ant staphylococcal antimicrobial treatment has been subject tinguish the S. aureus strains from other staphylococci: S.
to different iterations. The early strains of MRSA contained epidermidis. For this purpose, different biochemical tests
a complex cassette SCC mec containing several antimicro- were performed which are given below.
bial resistance genes when it appeared for the first time (Tay-
lor and Unakal 2017; John 2020). 2.3 Mannitol salt agar plates
Several antibiotics are used to treat the resistant strains
of S. aureus such as vancomycin but this antibiotic shows Samples were streaked on mannitol salt agar plate and
serious side effects like rashes at the site of injection and plates were kept at 37 ºC for overnight incubation. After
deafness (Chang et al. 2003). Plants constitute essential incubation, all plates were examined for S. aureus by colony
medication sources and alternative therapies since ancient morphology.
times to enhance symptoms and fight diseases. Today, due
to its possible effectiveness and lower side effects, the use 2.4 Baird‑parker medium
of plant related natural agents in medical care is drawings.
(Subramani et al. 2017). For the therapy against methicil- Streaked yellow colonies on Baird-parker medium and plates
lin resistant S. aureus infections, medicinal plants are being were kept at 37 ºC for overnight incubation. All plates were
used as remedies. This emphasizes a need for search of examined for S. aureus by colony morphology after over-
herbal extracts with anti-bacterial activity for the treatment night incubation.
of multidrug resistant S. aureus owing to the strong refer-
ence of use of medicinal plants as successful remedial agents 2.5 Hemolysis on blood agar
in various bacterial infections (Gonelimali et al. 2018).
The current study was an attempt to find out anti-bacterial Blood agar was used for studying hemolysis by the isolates.
activity of medicinal plant extracts against multidrug resist- Samples were streaked on blood agar plate and plates were
ant S. aureus. For this purpose, Local strains of S. aureus kept at 37 ºC for 18–24 h in aerobic atmosphere.
were identified from milk, nasal, blood, urine and pus sam-
ples. Drug susceptibility was identified though disc diffusion 2.6 Catalase test
method. Twenty-five medicinal plants were collected from
different regions of Saudi Arabia. They were air-dried and Catalase test was performed for differentiating S. aureus
extracted in methanol. These medicinal plants were screened from non-catalase producing bacteria. Briefly, the test was
for their anti-bacterial activity against multidrug resistant performed by adding bacterial cells from a colony to a drop
S. aureus. Half maximal inhibitory concentration ­(IC50) of of 3% hydrogen peroxide. Appearance of any bubbles was
these plant extracts was identified through micro-dilution noted.
method. Due to the promising results, it is hoped that our
plant extracts having efficacious anti-bacterial activities 2.7 Coagulase test
will prove to be quite useful when studied in more detail in
search of new therapeutic agents for better management of Staphylococcus aureus strains were further identified by
infections with multidrug resistant (MDR) S. aureus. positive bound coagulase test, which converts fibrinogens
directly to fibrin, using rabbit plasma. Briefly, the test was
performed by adding bacterial cells from a colony to rabbit
2 Materials and methods plasma in the test tube. Appearance of any coagulation was
noted.
2.1 Sample collection
2.8 Plant collection and extraction
Different samples (milk, nasal, urine, blood and pus) were
collected for isolation of S. aureus. Out of 47, 5 nasal sam- The plant material from 25 plants was collected from dif-
ples, 23 blood samples, 1 milk sample, 3 urine samples and ferent areas of Faisalabad on the basis of un-documented

13
 Rendiconti Lincei. Scienze Fisiche e Naturali

reports. The fresh plants or their parts were spread on 3 Results

filter paper and stored under shade at room temperature
(20–25 °C) for 7 days to avoid too many chemical changes 3.1 Positive S. aureus sample
for drying. The dried plants or their parts were chopped.
Then they were soaked in methanol and left overnight. The Forty-seven clinical and sub-clinical samples were collected
extracts were then filtered and fresh solvent now poured and from different areas of Faisalabad. Out of 47 samples, only
kept overnight with occasional shaking. The extract was fil- 21 (44.68%) were positive for S. aureus. Out of these, 2
tered on the following morning. Extracts of each plant was bacteria were obtained from nasal samples, 1 from blood
concentrated at temperature between 30 to 40 °C without samples, 1 from milk sample, 3 from urine samples and 14
bumping. Concentrated extracts were evaporated to dryness. was from pus sample.

2.9 Antimicrobial analysis of plants extracts

3.2 Biochemical test for S. aureus identification
The antimicrobial activity was determined on multidrug
3.2.1 Mannitol salt agar plates
resistant S. aureus. Standardized inoculums were prepared
and streaked as described in materials and methods of this
S. aureus uses mannitol as a nutrient and secretes an acid.
article. Sterile filter paper discs previously soaked in 10 μl
Phenol red is a pH indicator which detects the acid produc-
extract of known concentration were carefully placed at the
tion and yellow colonies appear, while other staphylococcus
center of the labeled plate. The plates were inverted and
species do not ferment mannitol and do not produce yel-
incubated aerobically at 37 °C in incubator and examined
low colonies. All samples were streaked on mannitol salt
for zones of inhibition after 19 h.
agar but only 21 exhibited yellow growth while others did
not show yellow growth on mannitol salt agar as shown in
2.10 Determination of half maximal inhibitory
Fig. 1.
concentration ­(IC50)

IC50 was determined by micro-dilution method using serially 3.2.2 

S. aureus growth
diluted (2 folds) plant extracts according to the Clinical and
Laboratory Standards Institute (CLSI). I­ C50 of the extracts For the growth of S. aureus, the carbon and nitrogen sources
was determined by twofolds dilution of plant extracts of con- are necessary. These sources are only present in the Baird-
centrations of 40 mg/ml in 10 test tubes. parker medium. Selective agents of this medium are lithium
chloride, glycine and potassium tellurite. S. aureus produces
2.11 Statistical analysis dark black shiny colonies due to reduction of tellurite. In the
present study mannitol confirmed 21 S. aureus were streaked
Statistical analysis was done by Graph pad prism software. on BP medium, and after 24 h, these 21 isolates exhibited
All values were written as mean ± SE. black colonies as depicted in Fig. 2.

Fig. 1  Growth of S. aureus

positive and negative

13
Rendiconti Lincei. Scienze Fisiche e Naturali

3.2.4 Catalase test

S. aureus produces an enzyme known as catalase that

decomposes hydrogen peroxide into water and oxygen as
appeared in Fig. 4.

3.2.5 Coagulase test

Staphylococcus aureus converts fibrinogens directly to

fibrin using rabbit plasma and shows clotting as shown in
the Fig. 5.

3.3 Antimicrobial resistance testing by disc

diffusion

Disc diffusion assay was performed on all S. aureus positive

samples. MHA was used for better diffusion of antibiotics
in antibiotic sensitivity test. The antimicrobial resistance
Fig. 2  S. aureus test by disc diffusion was performed on each isolate using 5
antibiotics. The overall resistance pattern of each antibiotic
was as follows: Oxacillin: 10 samples (47.6%), Ampicillin:
21 samples (100%), Chloramphenicol: 4 samples (19.04%),
Ciprofloxacin: 1 sample (4.76%) and Sulphamethoxazole: 5
samples (23.80%) as shown in Table 2.

3.4 Antimicrobial analysis of plants extracts

Plant materials selected on the basis of un-documented

reports for their anti-bacterial activity against S. aureus.
Different parts were collected and shade dried for a week.
Data of parts used and vernacular names were collected as
shown in Table 1.
Other extracts such as Withania coagulans, Artemisia
absinthium, Capsicum frutescens, Piper nigrum, Psidium
guajava, Nigella sativa, Acacia nilotica and Piper cubeba
extracts gave zone of inhibition as shown in data in Table 3.
Out of ten oxacillin resistant strains, three were tested
for ­IC50 against these five potent plant extracts: Rubia cor-
difolia, Punica granatum, Centratherum anthelminticum,
Lawsonia inermis and Eucalyptus globulus (Seeds) to find
out the 50% inhibition of S. aureus growth.
Fig. 3  Blood agar causing beta hemolysis The results of this research represented that the metha-
nolic extract of Centratherum anthelminticum gave 50%
inhibition of P7 strain of S. aureus at a concentration of
3.2.3 Hemolysis on blood agar 1.20 mg/ml, while P10 and P13 strains resulted in 50% inhi-
bition at a concentration of 1.20 mg/ml and 0.946 mg/ml
Blood agar is high-nutrient growth medium with 7% respectively.
sheep’s blood. It is a differential medium. It is used to In this experiment, the methanolic extract of Eucalyptus
differentiate closely related organisms such as S. aureus globulus (Seeds) gave I­ C50 of P7 and P10 strains of S. aureus
from other staphylococcal species. S. aureus produces beta at the concentration of 4.30 mg/ml and 3.38 mg/ml and P13
hemolysins that can be detected by their ability to com- resulted in 50% inhibition at a concentration of 4.43 mg/ml.
pletely lyse red blood cells. Figure 3. shows that S. aureus Methanolic extract of Lawsonia inermis exhibited ­IC50 of
causes beta-hemolysis in blood agar plate. P7 at a concentration of 2.07 mg/ml. while it showed 50%

13
 Rendiconti Lincei. Scienze Fisiche e Naturali

Fig. 4  Catalase test. Rapid, appearance of sustained gas bubbles by S. aureus by adding ­H2O2

The results of present study showed that the methanolic

extract of Rubia cordifolia, resulted in 50% inhibition of P7
and P10 S. aureus strain at a concentration of 0.280 mg/ml
each, while P13 gave ­IC50 at a concentration of 0.250 mg/
ml (Table 4).

4 Discussion

They are the source of causing broad range of infections

such as soft tissue infection, benign carriage, sepsis, bio-
film related prosthesis infection, superficial skin, deep
wound infections and life threatening bacteria. Being a most
opportunistic pathogen, it can cause more rigorous infec-
tions. These painstaking infections include a broad and wide
range such as carbuncle, boils, impetigo, deep lesions, furun-
cle, cellulites, osteomyelitis, chronic furunculosis boils,
abscesses, endocarditis and pneumonia. (Miller and Diep
2008). S. aureus is the clinically most significant by far, S.
aureus is present in 20–40% of the general population in
human commensal microbiota of nasal mucosa(Becker et al.
2017). When, for example, chronic skin disorders, wounds or
surgical operation damage cutaneous and mucosal defenses,
S. aureus may gain access to and cause infection. (Lowy
1998).
Continuously increasing drug resistance in S. aureus
Fig. 5  Coagulase test strains has aggravated the problem globally. Different strat-
egies are intended to restrict MRSA production through the
prudent use of antimicrobials, monitor the reservoir. MRSA
inhibition of P10 and P13 at a concentration of 1.99 mg/ml transmissions and infections are usually avoided by several
and 1.38 mg/ml. measures.
Methanolic extract of Punica granatum gave 50% inhibi- Staphylococcus aureus organized resistance against peni-
tion of P7 strain of S. aureus at a concentration of 0.530 mg/ cillin, methicillin, vancomycin, quinolone, tetracycline and
ml, while P10 and P13 strains resulted in 50% inhibition at erythromycin to evade the human immune system (Phougat
a concentration of 0.499 mg/ml each. et al. 2014).

13
Rendiconti Lincei. Scienze Fisiche e Naturali

Table 2  Multidrug resistant S. aureus identified by disc diffusion (mm)

Sample ID Oxacillin (1 µg) Ampicillin (10 µg) Chloramphe-nicol (30 µg) Ciprofloxacin (5 µg) Sulphameth-
oxazole (25 µg)

P1 15.67 ± 0.33 S 8.33 ± 0.88 R 23.00 ± 0.57 S 22.67 ± 0.88 S 8.00 ± 0.57 R
P2 19.33 ± 0.88 S 12.00 ± 0.57 R 21.67 ± 0.88 S 25.00 ± 0.57 S 9.00 ± 0.57 R
P3 15.00 ± 0.57 S 25.67 ± 0.88 R 29.67 ± 0.88 S 32.33 ± 0.88 S 30.67 ± 0.88 S
P5 7.33 ± 0.33 R 8.00 ± 0.57 R 8.00 ± 0.00 R 24.00 ± 0.57 S 22.17 ± 0.60 S
P7 0.00 ± 0.00 R 8.33 ± 0.88 R 9.33 ± 0.88 R 22.33 ± 0.88 S 25.33 ± 0.88 S
P8 15.17 ± 0.44 S 26.83 ± 0.92 R 19.00 ± 0.57 S 34.33 ± 0.88 S 36.17 ± 0.60 S
P9 6.00 ± 0.00 R 15.00 ± 0.57 R 22.67 ± 0.88 S 26.83 ± 0.92 S 24.33 ± 0.44 S
P10 0.00 ± 0.00 R 7.00 ± 0.57 R 26.50 ± 0.28 S 34.00 ± 0.57 S 27.17 ± 0.44 S
P11 4.00 ± 2.00 R 7.16 ± 0.60 R 27.83 ± 0.44 S 34.17 ± 0.16 S 26.50 ± 0.28 S
P12 4.00 ± 2.00 R 0.00 ± 0.00 R 13.17 ± 0.44 I 23.50 ± 0.28 S 15.17 ± 0.44 S
P13 0.00 ± 0.00 R 0.00 ± 0.00 R 21.17 ± 0.72 S 26.33 ± 0.33 S 26.50 ± 0.28 S
P14 14.83 ± 0.16 S 9.00 ± 0.57 R 23.50 ± 0.28 S 24.17 ± 0.44 S 19.17 ± 0.60 S
P15 4.00 ± 2.00 R 0.00 ± 0.00 R 11.83 ± 0.44 R 12.50 ± 0.28 R 20.00 ± 0.57 S
U1 15.17 ± 0.44 S 8.16 ± 0.44 R 22.00 ± 0.57 S 22.50 ± 0.28 S 7.00 ± 0.57 R
U2 17.57 ± 0.28 S 7.50 ± 0.76 R 21.67 ± 0.33 S 23.00 ± 0.57 S 7.00 ± 0.57 R
U3 14.83 ± 0.16 S 8.16 ± 0.44 R 21.00 ± 0.57 S 23.00 ± 0.57 S 6.66 ± 0.33 R
N1 7.33 ± 0.66 R 10.00 ± 0.57 R 21.83 ± 0.44 S 21.67 ± 0.33 S 28.00 ± 0.28 S
N2 6.66 ± 0.66 R 9.00 ± 0.57 R 12.83 ± 0.44 R 22.17 ± 0.16 S 24.17 ± 0.60 S
N3 15.17 ± 0.44 S 21.00 ± 0.57 R 21.50 ± 0.76 S 30.50 ± 0.28 S 32.17 ± 0.44 S
M1 15.00 ± 0.57 S 20.00 ± 0.57 R 21.67 ± 0.33 S 23.17 ± 0.60 S 27.83 ± 0.72 S
B1 17.50 ± 0.28 S 10.17 ± 0.44 R 21.83 ± 0.60 S 21.50 ± 0.28 S 19.00 ± 0.57 S

Treatment of S. aureus infections rest on largely on the ethanol, chloroform, petroleum ether, acetone, alcohol and
category of infection as well as the occurrence or absence ethyl acetate are being extracted from different part of the
of drug resistant strains. When antimicrobial therapy is plant such as seed, leaves, fruit, root, bark, stem and whole
required, the duration and mode of therapy are mostly reli- plant. These extracts have been tested against various micro-
ant on the infection type as well as other factors.(Tong et al. bial infections and have given efficient results. Therefore
2015). it seems necessary to search new antimicrobial substances
Staphylococcus aureus is increasingly developing resist- from medicinal plants that can not only target microbial
ance to all classes of antibiotics (Amyes and Young 2014). infections but also can work efficiently against multidrug
In present study, 47.6% isolates gave oxacillin resistance, resistance pathogens (Munuswamy et al. 2013).
23.80% were resistant to Sulphamethoxazole, 19.04% were From twenty five, 5 medicinal plants show anti-bacterial
resistant to chloramphenicol, 4.76% were resistant to cipro- activity against S. aureus. Other extracts such as Withania
floxacin while all were resistant to ampicillin. Resistance to coagulans, Artemisia absinthium, Capsicum frutescens,
Oxacillin and other drugs was confirmed by disc diffusion Piper nigrum, Psidium guajava, Nigella sativa, Acacia nilot-
assay (Sorour et al. 2014). ica and Piper cubeba show anti-bacterial activity (Table 3).
As the S. aureus is increasingly developing resist- Centratherum anthelminticum commonly known as Kali-
ance to all classes of antibiotics therefore, there is a need giri. It is used as treatment of leucoderma and other skin
to develop strategies that can solve the problem of resist- diseases. Its seeds are used as purgative, kidney dilemma,
ance in S. aureus. For the treatment of methicillin resist- useful in inflammatory swelling, for asthma. Organic and
ant S. aureus infections, medicinal plants are being used as aqueous extracts of these seeds were scientifically exam-
remedies(Okwu et al. 2019). ined for anti-bacterial, larvicidal, anti-fungal, anticancer,
For this reason, information about traditional usage of anti-oxidant, anthelmintic, anti-diabetic, analgesic, antipy-
potent component/s of plants for medicinal purposes, their retic, anti-viral, anti-inflammatory, diuretic, wound healing
scientific names and local names was collected (Table 1). activities (Paydar et al. 2013). There is no scientific evi-
Medicinal plants have always been used against micro- dence regarding drug resistance of S. aureus against this
bial infections due to their low cost and phytochemicals that plant. Based on the above fact, the anti-bacterial activity of
have antimicrobial activity. Different solvents like methanol, Centratherum anthelminticum was evaluated using in vitro

13
13
Table 3  Zone of inhibition of plant extracts (mm) against multidrug resistant S. aureus
Plant extracts Samples IDs of bacteria
P5 P7 P9 P10 P11 P12 P13 P15 N1 N2

Acacia nilotica (Beans) 13.67 ± 0.44 16.33 ± 0.88 15.17 ± 0.72 15.50 ± 0.28 14.83 ± 0.44 16.33 ± 0.88 17.33 ± 0.88 19.00 ± 0.57 17.50 ± 0.76 15.50 ± 0.28
Acacia nilotica (Leaves) 15.17 ± 0.72 16.83 ± 0.44 14.83 ± 0.44 12.00 ± 0.57 10.17 ± 0.44 17.50 ± 0.76 17.17 ± 0.44 17.33 ± 0.88 17.17 ± 0.44 16.83 ± 0.44
Acacia suma 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Aloe vera 10.17 ± 0.44 12.50 ± 0.28 12.00 ± 0.57 12.33 ± 0.88 9.83 ± 0.44 12.50 ± 0.28 8.50 ± 0.28 11.83 ± 0.44 8.83 ± 0.44 10.17 ± 0.44
Artemisia absinthiu-m 12.33 ± 0.88 15.50 ± 0.28 9.16 ± 0.44 13.50 ± 0.28 10.50 ± 0.28 12.00 ± 0.57 15.17 ± 0.72 8.83 ± 0.44 14.83 ± 0.44 11.83 ± 0.44
Berberis lycium 9.16 ± 0.44 10.17 ± 0.44 8.83 ± 0.44 8.50 ± 0.28 7.83 ± 0.44 9.83 ± 0.44 8.00 ± 0.57 8.50 ± 0.28 9.16 ± 0.44 7.83 ± 0.44
Capsicum frutescens 10.50 ± 0.28 12.50 ± 0.28 11.83 ± 0.44 12.33 ± 0.88 11.83 ± 0.44 13.67 ± 0.44 8.83 ± 0.44 9.16 ± 0.44 12.00 ± 0.57 10.50 ± 0.28
Centrathe-rum anthelmin-ticum 17.33 ± 0.88 16.33 ± 0.88 14.83 ± 0.44 14.83 ± 0.44 8.50 ± 0.28 9.83 ± 0.44 20.33 ± 0.72 18.83 ± 0.44 17.50 ± 0.76 16.83 ± 0.44
Citrullus colocynthis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Eucalyptus globulus (Leaves) 11.83 ± 0.44 8.50 ± 0.28 10.17 ± 0.44 12.50 ± 0.28 12.00 ± 0.57 12.33 ± 0.88 9.83 ± 0.44 12.50 ± 0.28 11.83 ± 0.44 8.83 ± 0.44
Eucalyptus globulus (Seeds) 9.16 ± 0.44 15.50 ± 0.28 13.50 ± 0.28 13.67 ± 0.44 8.83 ± 0.44 15.17 ± 0.72 16.83 ± 0.44 12.33 ± 0.88 16.33 ± 0.88 17.33 ± 0.88
Fumaria officinalis 12.00 ± 0.57 12.50 ± 0.28 9.83 ± 0.44 10.50 ± 0.28 8.50 ± 0.28 10.17 ± 0.44 8.83 ± 0.44 8.50 ± 0.28 9.16 ± 0.44 11.83 ± 0.44
Lawsonia inermis 15.50 ± 0.28 16.83 ± 0.44 13.67 ± 0.44 15.50 ± 0.28 8.83 ± 0.44 15.17 ± 0.72 22.50 ± 0.28 17.50 ± 0.76 16.33 ± 0.88 17.33 ± 0.88
Moringa oleifera 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Nigella sativa 12.00 ± 0.57 13.67 ± 0.44 9.83 ± 0.44 15.17 ± 0.72 12.33 ± 0.88 12.50 ± 0.28 16.33 ± 0.88 14.83 ± 0.44 15.50 ± 0.28 14.83 ± 0.44
Peganum harmala 12.50 ± 0.28 11.83 ± 0.44 12.33 ± 0.88 9.16 ± 0.44 12.50 ± 0.28 10.50 ± 0.28 9.83 ± 0.44 9.16 ± 0.44 12.33 ± 0.88 8.50 ± 0.28
Piper cubeba 11.83 ± 0.44 10.50 ± 0.28 9.16 ± 0.44 15.17 ± 0.72 12.33 ± 0.88 12.50 ± 0.28 13.67 ± 0.44 10.17 ± 0.44 12.50 ± 0.28 12.00 ± 0.57
Piper nigrum 12.00 ± 0.57 9.83 ± 0.44 10.17 ± 0.44 12.50 ± 0.28 11.83 ± 0.44 12.00 ± 0.57 10.50 ± 0.28 12.50 ± 0.28 11.83 ± 0.44 13.50 ± 0.28
Psidium guajava 12.50 ± 0.28 14.83 ± 0.44 13.67 ± 0.44 12.33 ± 0.88 12.00 ± 0.57 13.50 ± 0.28 11.83 ± 0.44 15.17 ± 0.72 13.67 ± 0.44 12.50 ± 0.28
Punica granatum 18.83 ± 0.44 16.33 ± 0.88 17.33 ± 0.88 19.00 ± 0.57 21.00 ± 0.57 16.83 ± 0.44 23.83 ± 0.16 18.83 ± 0.44 20.33 ± 0.72 17.50 ± 0.76
Rubia cordifolia 16.83 ± 0.44 17.50 ± 0.76 12.50 ± 0.28 15.50 ± 0.28 18.83 ± 0.44 15.17 ± 0.72 19.00 ± 0.57 17.17 ± 0.44 18.83 ± 0.44 17.33 ± 0.88
Santalum album 12.00 ± 0.57 12.50 ± 0.28 11.83 ± 0.44 8.83 ± 0.44 9.83 ± 0.44 12.00 ± 0.57 8.50 ± 0.28 10.50 ± 0.28 12.33 ± 0.88 11.83 ± 0.44
Solanum nigrum 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Sphaerant-hus indicus 9.83 ± 0.44 10.17 ± 0.44 8.83 ± 0.44 8.50 ± 0.28 9.83 ± 0.44 12.00 ± 0.57 9.16 ± 0.44 8.50 ± 0.28 9.16 ± 0.44 11.83 ± 0.44
Swertia chirata 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Syzygium cumini 8.83 ± 0.44 10.17 ± 0.44 12.50 ± 0.28 12.00 ± 0.57 12.50 ± 0.28 11.83 ± 0.44 8.50 ± 0.28 8.83 ± 0.44 10.50 ± 0.28 12.33 ± 0.88
Withania coagulans 15.50 ± 0.28 16.83 ± 0.44 19.00 ± 0.57 17.17 ± 0.44 17.33 ± 0.88 16.33 ± 0.88 18.83 ± 0.44 19.00 ± 0.57 17.50 ± 0.76 15.50 ± 0.28
Rendiconti Lincei. Scienze Fisiche e Naturali
Rendiconti Lincei. Scienze Fisiche e Naturali

Table 4  IC50 values of S. aureus isolates

Sample IDs of Centratherum anthel- Eucalyptus globulus Lawsonia inermis Punica granatum Rubia cordifolia
bacteria minticum
IC50 (mg/ml) IC50 (mg/ml) IC50 (mg/ml) IC50 (mg/ml) IC50 (mg/ml)

P7 1.02 4.30 2.07 0.530 0.280

P10 1.20 3.38 1.99 0.499 0.280
P13 0.946 4.43 1.38 0.499 0.250

Fig. 6  A Oxacillin, B ampicillin, C chloramphenicol, D ciprofloxa- Fig. 7  Effect of medicinal plants on multidrug resistant S. aureus
cin, E Sulphamethoxazole (Isolate #). RC Rubia cordifolia, LI Lawsonia inermis, PG Punica
granatum, EG Eucalyptus globulus, CA Centratherum anthelmenticus

experimental isolates of S. aureus from clinical and sub-

clinical samples. Centratherum anthelminticum seeds was 5 Results
studied using S. aureus isolated from clinical and sub-clin-
ical samples. The antimicrobial activity of Centratherum Pharmacological activities of the leaf extract of Lawsonia
anthelminticum showed the inhibitory effect at a concentra- inermis include sedative and nootropic effects (Ahmed and
tion of 40 µg/ml on all three multidrug resistant strains of S. Javed 2014). Sukanya et al. (2009) described that 6 mm
aureus isolated from clinical and sub-clinical samples i.e., zone of inhibitory activity by leaves of Lawsonia inermis
P7, P10 and P13. The zone of inhibition for clinical iso- against clinical S. aureus was observed with well dilution
lates of S. aureus i.e., P7, P10 and P13 was 15 mm, 16 mm method (Sukanya et al. 2009). Conclusion of antimicrobial
and 19 mm respectively. Figure of extract of Centratherum activity showed extract of Lawsonia inermis. The zone
anthelminticum is 0.946–1.20 (see Fig. 6). of inhibition for clinically isolated S. aureus i.e., P7, P10
The Eucalyptus globulus is used to control wide range and P13 was 16 mm, 15 mm and 22 mm respectively. It
of diseases derived from bacterial infections (Ghalem and exhibited ­IC50 value at a concentration between 1.38 and
Mohamed 2008). Though, only limited information is 2.07 mg/ml.
accessible so far regarding its effectiveness. Present study Punica granatum rind has a good deal of antimicrobial
revealed that isolates of multidrug resistant S. aureus were activity (Al-Zoreky 2009). The rind is used as an astrin-
absolutely inhibited at 40 µg/ml. The zone of inhibition for gent (Prashanth et al. 2001). Khan et al. (2014) described
clinical isolated S. aureus i.e., P7, P10 and P13 was 15 mm, that the methanolic extract of fruit peels of P. granatum
13 mm and 16 mm respectively and gave ­IC50 value between was also effective against S. aureus (Khan et al. 2014). Our
3.38 and 4.43 mg/ml (see Fig. 7).

13
 Rendiconti Lincei. Scienze Fisiche e Naturali

study was thus planned to evaluate this characteristic of P. Data availability The authors acknowledge that all relevant data pre-
granatum. The outcomes of disc diffusion assay verified sented in this study are contained within the article, further inquiries
can be directed to the corresponding authors.
that isolates of multi-drug resistant S. aureus were totally
inhibited on 6 mm disc. Zone for clinical isolates of S. Declarations
aureus i.e., P7, P10 and P13 was 15 mm, 18 mm and 24.
Rubia cordifolia is a significant plant which is valuable Conflicts of interest No conflict of interest exists in the submission of this
for cure of range of diseases. It also has been found to hinder manuscript, and manuscript is approved by all authors for publication.
lipid peroxidation to the best of our knowledge this plant has
never been used as antimicrobial agent against S. aureus. In
our study, we used methanolic extract of root of Rubia cordi- References
folia against oxacillin and other drug resistant S. aureus and
Ahmed S, Javed I (2014) Anxiolytic effect of Lawsonia inermis Linn
it produces quite large zones. Zone for clinically isolated S. (Hena) on light dark box activity. World J Pharm Pharm Sci
aureus i.e., P7, P10 and P13 was 17 mm, 15 mm and 18 mm, 3(5):1334–1339
showed ­IC50 figures 0.250–0.280. Al-Zoreky N (2009) Antimicrobial activity of pomegranate (Punica
granatum) fruit peels. Int J Food Microbiol 134(3):244–248
Keeping in view the anti-bacterial progress of the extracts
Amyes S, Young H (2014) Antibiotic resistance: theory into practice.
of Centratherum anthelminticum (seeds), Eucalyptus glob- Encycl Genet 125
ules (seeds), Lawsonia inermis (leaves), Punica granatum Becker K, Schaumburg F, Fegeler C, Friedrich AW, Köck R (2017)
(peel), Rubia cordifolia (root) opposition of different S. Staphylococcus aureus from the German general population is
highly diverse. Int J Med Microbiol 307(1):21–27
aureus isolates as explored in this study, it is anticipated
Boucher HW, Corey GR (2008) Epidemiology of methicil-
that these plants will lead character in the treatment of S. lin-resistant Staphylococcus aureus. Clin Infect Dis
aureus infections. 46(Supplement_5):S344–S349
Calfee D, Salgado C, Milstone A, Harris A, Kuhar D, Moody J,
Aureden K, Huang S, Maragakis L, Yodoe D (2014) Society for
Healthcare Epidemiology of America. Strategies to prevent methi-
cillin-resistant Staphylococcus aureus transmission and infection
6 Conclusion in acute care hospitals: 2014 update. Infect Control Hosp Epide-
miol 35:772–796
This article has reported the resistance of different drugs Chang S, Sievert DM, Hageman JC, Boulton ML, Tenover FC, Downes
against S. aureus as well as investigated the activities of FP, Shah S, Rudrik JT, Pupp GR, Brown WJ (2003) Infection with
vancomycin-resistant Staphylococcus aureus containing the vanA
medicinal plants against S. aureus. These data also recom- resistance gene. N Engl J Med 348(14):1342–1347
mend a strong opportunity of these extracts being used as Galani VJ, Panchal RR (2014). Invitro evaluation of Centratherum
drugs. It is hoped that this work will further help to find out anthelminticum seeds for antinephrolithiatic activity. J Homeopa-
active ingredients from plant extracts which shows potent thy Ayurv Med
Ghalem BR, Mohamed B (2008) Antibacterial activity of leaf essential
anti-bacterial effect against MRSA which is a threatening oils of Eucalyptus globulus and Eucalyptus camaldulensis. Afr J
pathogen. The active ingredients will be used for drug devel- Pharm Pharmacol 2(10):211–215
opment against MRSA. Gonelimali FD, Lin J, Miao W, Xuan J, Charles F, Chen M, Hatab SR
(2018) Antimicrobial properties and mechanism of action of some
Acknowledgements Princess Nourah bint Abdulrahman University plant extracts against food pathogens and spoilage microorgan-
Researchers Supporting Project number (PNURSP2022R148), Princess isms. Front Microbiol 9:1639
Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. Guerrini A, Mancini I, Maietti S, Rossi D, Poli F, Sacchetti G, Gam-
bari R, Borgatti M (2014) Expression of pro-inflammatory
Author contributions RA: conceptualization, data curation, formal interleukin-8 is reduced by ayurvedic decoctions. Phytother Res
analysis, investigation, methodology, software, validation, writing— 28:1173–1181
original draft, writing—review & editing; TSA: conceptualization, Jayasundara NS (2014) An investigation of Staphylococcus aureus
data curation, formal analysis, investigation, methodology, software, and related species from flood affected and other environmental
validation, writing—original draft, writing—review & editing; WAA, sources. Queensland University of Technology
AAH: conceptualization, data curation, formal analysis, investigation, Joharapurkar A, Deode N, Zambad S, Umathe S (2003) Immunomodu-
methodology, software, validation, writing—original draft, writing— latory activity of alcoholic extract of Rubia cordifolia Linn. Indian
review & editing.: conceptualization, data curation, formal analysis, Drugs 40(3):179–181
methodology, software, validation, writing—review & editing; RAA, John J Jr (2020) The treatment of resistant staphylococcal infections.
ASMA, NA: data curation, formal analysis methodology, visualiza- F1000Research 9:F1000 Faculty Rev-150
tion; AA, KSA, SSA, AAA: conceptualization, data curation, formal Kasture VS, Deshmukh V, Chopde C (2000) Anticonvulsant and behav-
analysis, methodology, software, validation software, methodology, ioral actions of triterpene isolated from Rubia cordifolia Linn.
visualization; MSA, BsMA, HMA: conceptualization, data curation, Indian J Exp Biol 38(7):675–680
formal analysis, methodology, software, methodology, visualization; Khan N, Abbasi AM, Dastagir G, Nazir A, Shah GM, Shah MM, Shah
NAA: conceptualization, funding acquisition, project administra- MH (2014) Ethnobotanical and antimicrobial study of some
tion, resources, supervision, visualization, writing—review & edit- selected medicinal plants used in Khyber Pakhtunkhwa (KPK) as
ing. All authors have read and agreed to the published version of the a potential source to cure infectious diseases. BMC Complement
manuscript. Altern Med 14(1):122

13
Rendiconti Lincei. Scienze Fisiche e Naturali

Lee AS, de Lencastre H, Garau J, Kluytmans J, Malhotra-Kumar S, Sorour AE, El-Awady BA, Mukhtar A (2014) Co-existance of Ami-
Peschel A, Harbarth S (2018) Methicillin-resistant Staphylococcus noglycoside Modifying Enzymes (AMEs) genes and mec-A gene
aureus. Nat Rev Dis Primers 4(1):18033 among nosocomial isolates of Staphylococcus aureus in Surgical
Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med Intensive Care Units in Kasr Al-Ainy hospitals, Cairo University.
339(8):520–532 Int Arab J Antimicrob Agents 3(4)
Mastoraki A, Kriaras I, Douka E, Mastoraki S, Stravopodis G, Gerou- Stryjewski ME, Corey GR (2014) Methicillin-resistant Staphylococcus
lanos S (2008) Methicillin-resistant Staphylococcus aureus pre- aureus: an evolving pathogen. Clin Infect Dis 58(suppl 1):S10–S19
venting strategy in cardiac surgery. Interact Cardiovasc Thorac Subramani R, Narayanasamy M, Feussner K-D (2017) Plant-derived
Surg 7(3):452–456 antimicrobials to fight against multi-drug-resistant human patho-
Miller LG, Diep BA (2008) Colonization, fomites, and virulence: rethink- gens. 3 Biotech 7(3):172
ing the pathogenesis of community-associated methicillin-resistant Sukanya S, Sudisha J, Hariprasad P, Niranjana S, Prakash H, Fathima S
Staphylococcus aureus infection. Clin Infect Dis 46(5):752–760 (2009) Antimicrobial activity of leaf extracts of Indian medicinal plants
Munuswamy H, Thirunavukkarasu T, Rajamani S, Elumalai EK, Ernest against clinical and phytopathogenic bacteria Afr J Biotechnol 8(23)
D (2013) A review on antimicrobial efficacy of some traditional Taylor TA, Unakal CG (2017) Staphylococcus Aureus. StatPearls Pub-
medicinal plants in Tamil Nadu. J Acute Dis 2(2):99–105 lishing, Treasure Island, FL, USA
Okwu MU, Olley M, Akpoka AO, Izevbuwa OE (2019) Methicillin- Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG (2015) Staphy-
resistant Staphylococcus aureus (MRSA) and anti-MRSA activi- lococcus aureus infections: epidemiology, pathophysiology, clinical
ties of extracts of some medicinal plants: a brief review. AIMS manifestations, and management. Clin Microbiol Rev 28(3):603–661
Microbiol 5(2):117 Turner NA, Sharma-Kuinkel BK, Maskarinec SA, Eichenberger EM,
Paydar M, Moharam BA, Wong YL, Looi CY, Wong WF, Nyamathulla S, Shah PP, Carugati M, Holland TL, Fowler VG (2019) Methicillin-
Pandy V, Kamalidehghan B, Arya A (2013) Centratherum anthelmin- resistant Staphylococcus aureus: an overview of basic and clinical
ticum (L.) Kuntze a potential medicinal plant with pleiotropic pharma- research. Nat Rev Microbiol 17(4):203–218
cological and biological activities. Int J Pharmacol 9(3)
Phougat N, Khatri S, Singh A, Dangi M, Kumar M, Dabur R, Kumar Publisher's Note Springer Nature remains neutral with regard to
Chhillar A (2014) Combination Therapy: the propitious ration- jurisdictional claims in published maps and institutional affiliations.
ale for drug development. Comb Chem High Throughput Screen
17(1):53–67 Springer Nature or its licensor (e.g. a society or other partner) holds
Prashanth D, Asha M, Amit A (2001) Antibacterial activity of Punica exclusive rights to this article under a publishing agreement with the
granatum. Fitoterapia 72(2):171–173 author(s) or other rightsholder(s); author self-archiving of the accepted
Rao GMM, Rao CV, Pushpangadan P, Shirwaikar A (2006) Hepatopro- manuscript version of this article is solely governed by the terms of
tective effects of rubiadin, a major constituent of Rubia cordifolia such publishing agreement and applicable law.
Linn. J Ethnopharmacol 103(3):484–490

Authors and Affiliations

Rasha Assiri1 · Nada Abdullah Alharbi1,13 · Thamir Saad Alsaeed2 · Waleed Al Abdulmonem2 ·
Almonther Abdullah Hershan3 · Rana Abdullah Alghamdi4 · Abdullah S. M. Aljohani5 · Nada Alkhorayef6 ·
Ahmad Almatroudi7 · Khaled S. Allemailem7 · Samia S. Alkhalil8 · Ameena A. AL‑surhanee9 · Mariam S. Al‑Ghamdi10 ·
Basal Sulaiman M. Alkhudhairy11 · Hailah M. Almohaimeed12

1 8
Department of Basic Medical Sciences, College Department of Clinical Laboratory Sciences, Faculty
of Medicine, Princess Nourah bint Abdulrahman University, of Applied Medical Sciences, Shaqra University, Shaqra,
P.O. Box 11671, Riyadh, Saudi Arabia Saudi Arabia
2 9
Department of Pathology, College of Medicine, Qassim Biology Department, College of Science, Jouf University,
University, P.O. Box 6655, Buraidah 51452, Qassim, Sakaka 2014, Saudi Arabia
Saudi Arabia 10
Department of Biology, College of Applied Sciences, Umm
3
Department of Medical Microbiology and Parasitology, Al-Qura University, Mecca, Saudi Arabia
College of Medicine, The University of Jeddah, Jeddah, 11
College of Medicine, Shaqra University, P.O. Box 13343,
Saudi Arabia
Riyadh 7396, Saudi Arabia
4
Department of Chemistry, College of Sciences and Arts, 12
Department of Basic Science, College of Medicine, Princess
King Abdulaziz University, Rabigh, Saudi Arabia
Nourah bint Abdulrahman University, P.O. Box 84428,
5
Department of Veterinary Medicine, College of Agriculture Riyadh, Saudi Arabia
and Veterinary Medicine, Qassim University, Buraydah, 13
Department of Basic Medical Sciences, Unaizah College
Saudi Arabia
of Medicine and Medical Sciences, Qassim University,
6
Department of Clinical Laboratory Science, College Qassim, Saudi Arabia
of Applied Medical Sciences, Al‑Quway’iyah, Shaqra
University, Riyadh, Saudi Arabia
7
Department of Medical Laboratories, College of Applied
Medical Sciences, Qassim University, Buraydah 51452,
Saudi Arabia

13