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Original Research Article
Antimicrobial activity of Cannabis sativa,Thuja orientalis and Psidium
guajava leaf extracts against methicillin-resistant Staphylococcus aureus
Shohini Chakraborty
a
, Nashra Afaq
a
, Neelam Singh
a
, Sukanta Majumdar
b,
⇑
a
Department of Microbiology, Saaii College of Medical Science and Technology, Chaubepur, Kanpur 209203, India
b
Microbiology and Microbial Biotechnology Laboratory, Department of Botany, University of Gour Banga, Malda, West Bengal 732103, India
article info
Article history:
Received 31 January 2018
Accepted 9 June 2018
Available online 29 July 2018
Keywords:
Methicillin-resistant Staphylococcus aureus
Antimicrobial activity
Plant extracts
Synergistic effect
High-performance thin-layer
chromatography
abstract
Objective: This study examined the antimicrobial activity of Cannabis sativa,Thuja orientalis and Psidium
guajava against methicillin-resistant Staphylococcus aureus (MRSA) and used a standardized purification
protocol to determine the presence and abundance of bioactive compounds in the leaf extracts.
Methods: In vitro antimicrobial activities of the ethanolic extracts of C. sativa,T. orientalis and P. guajava
were tested against MRSA. The presence of bioactive molecules in these three leaves was evaluated using
biochemical assays and high-performance thin-layer chromatography (HPTLC).
Results: Resistance to methicillin, penicillin, oxacillin and cefoxitin was observed in each of the clinical
and nonclinical MRSA isolates. However, they were still vulnerable to vancomycin. Used individually,
the 50% extract of each plant leaf inhibited MRSA growth. A profound synergism was observed when
C. sativa was used in combination with T. orientalis (1:1) and when P. guajava was used in combination
with T. orientalis (1:1). This was shown by larger zones of inhibition. This synergism was probably due
to the combined inhibitory effect of phenolics present in the leaf extracts (i.e., quercetin and gallic acid)
and catechin, as detected by HPTLC.
Conclusion: The leaf extracts of C. sativa,T. orientalis and P. guajava had potential for the control of both
hospital- and community-acquired MRSA. Moreover, the inhibitory effect was enhanced when extracts
were used in combination.
Please cite this article as: Chakraborty S, Afaq N, Singh N, Majumdar S. Antimicrobial activity of Cannabis
sativa,Thuja orientalis and Psidium guajava leaf extracts against methicillin-resistant Staphylococcus aur-
eus.J Integr Med. 2018; 16(5): 350–357.
Ó2018 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.
1. Introduction
Staphylococcus aureus is known to cause wide spectrum of pyo-
genic lesions, involving several organs, and hospital-acquired and
community-acquired infections are both well recognized. In
nature, S. aureus infections are often fatal. Some strains have
developed resistance to several b-lactam antibiotics used in hospi-
tals. Methicillin-resistant S. aureus (MRSA) is a major opportunistic
pathogen that causes both nosocomial and community-acquired
infections (community-associated MRSA, CA-MRSA) [1].S. aureus
is a Gram-positive, coagulase-positive coccus of the family of
Staphylococcaceae. Since it was first identified in 1961, MRSA
has spread throughout the world and become one of the most
frequent pathogenic bacterium causing healthcare-associated
infections. MRSA can cause various types of life-threatening infec-
tions, such as septic shock, endocarditis and severe pneumonia
[2–5]. Different MRSA strains have emerged worldwide, and they
have become resistant to a variety of antibiotics, including
penicillin, tetracycline, methicillin and vancomycin. Recently, in
several countries, MRSA has been found to infect livestock and
the humans exposed to those infected animals. This type of MRSA
has been named livestock-associated MRSA (LA-MRSA) [6]. The
interactions among these different types of MRSA reservoirs have
been reported, including nosocomial infections by CA-MRSA and
importation of LA-MRSA in hospitals, leading to hospital-acquired
infections [7–9]. As a result of inadequate hygiene procedures in
a variety of settings, the transmission of resistant bacteria (e.g.,
MRSA) from farm animals to humans has been reported to occur
not only by physical contact with the animals, but also through
consumption of meat or other food products (e.g., beef and dairy
food) [10,11].S. aureus is common in foods and food-related
https://doi.org/10.1016/j.joim.2018.07.005
2095-4964/Ó2018 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.
⇑
Corresponding author.
E-mail address: smajumdarwbes@gmail.com (S. Majumdar).
Journal of Integrative Medicine 16 (2018) 350–357
Contents lists available at ScienceDirect
Journal of Integrative Medicine
journal homepage: www.jcimjournal.com/jim
www.journals.elsevier.com/journal-of-integrative-medicine
environments (e.g., kitchen and food retail spaces). MRSA may fol-
low the same infection transmission pattern in raw food, cooked
food products (like meat, fish), and among human beings
[12–14]. Thus, meals prepared in public hospitals and food retail
spaces should receive special attention, because these foods are
intended for consumption by patients, a population at high risk
of acquiring various diseases caused by S. aureus [15]. MRSA, due
to a modification in penicillin-binding protein 2a, has a decreased
affinity to b-lactam. The mecA gene encodes this protein and is
located on a mobile SCCmec cassette chromosome. This genetic
element resists to most currently available b-lactam antibiotics.
Unfortunately, though several agents and protocols have been pro-
posed, no prophylactic strategies have yet been proven useful.
Therefore, new alternative therapies have to be developed to kill
extended-spectrum b-lactamase (ESBL) strains of MRSA. Herbal
medicine can solve this problem, and many plant extracts have
been reported to possess inhibitory activity towards S. aureus as
well as MRSA and ESBL-MRSA.
Medicinal plants, such as Psidium guajava L., Rosmarinus
officinalis L., Salvia fruticosa Mill, Majorana syriaca (L.) Raf., Ocimum
basilicum L., Syzygium aromaticum (L.) Merr. & L.M. Perry, Laurus
nobilis L. and Rosa damascena Herrm., have been shown to enhance
the effects of known antibiotics, such as oxytetracycline, gen-
tamycin, penicillin, enrofloxacin and cephalexin, against clinical
isolates of S. aureus. The synergistic effects of the combination of
multiple extracts against MRSA strains showed more effectiveness
than strains without antibiotic resistance (14 mm zone for
methicillin is considered resistant) [16].
An in vitro study evaluated the synergistic effects of traditional
medicinal plants from the south-west of Nigeria for the treatment
of Salmonella typhi infections. In this study, combinations of plant
extracts were tested, but no manufactured antibiotics were used.
Different combinations of extracts of 2–9 plants (in methanol or
water) were tested against a pathogenic bacterium and were found
to have strong effects. The plants used in this study included Carica
papaya,Citrus aurantifolia,Ananas sativus,C. paradisi,Cymbopogon
citratus (lemon grass), Cocos nucifera,Euphorbia heterophylla and
Gossypium spp. The authors found that the most effective synergis-
tic combinations were: (1) C. papaya +C. aurantifolia with 16 mm
clearing (individually 10 mm and 11 mm); (2) C. papaya +C. auran-
tifolia +Gossypium +C. nucifera +C. paradisi, with 27 mm clearing
(individually 10–12 mm only) [17].
The synergy between some common plant extracts and antibi-
otics has been reported by many scientists. A pronounced synergis-
tic effect was observed when S. officinalis and Foeniculum vulgare
were used against S. aureus,Escherichia coli,Bacillus subtilis and
Listeria monocytogenes [18–22]. Some authors tested the antimi-
crobial activity and synergism in crude extracts and essential oils
obtained from plants such as S. officinalis and S. sclera. The essential
oils from S. officinalis with oxacillin exhibited clear synergistic
effect against S. epidermidis [23]. Extracts of Mimosa pudica,Ixora
coccinea,Colocasia esculenta and Boerhavia diffusa did not show
strong antibacterial activity against MRSA when tested individu-
ally. However, when extract of C. esculenta was given in combina-
tion with gentamycin, a tenfold increase in the activity of
gentamycin was noticed [24]. The present study was conducted
to evaluate the anti-MRSA effects of ethanolic extracts of Psidium
guajava,Thuja orientalis and Cannabis sativa, as well as the syner-
gistic effects of using them in combination.
2. Materials and methods
2.1. Isolation and identification of microbial strains
Both clinical (10) samples and nonclinical (10) specimens were
collected during a one-year period (2015–2016). The total 20 swab
samples were streaked on mannitol salt agar (MSA) media (HiMedia
Laboratories, Mumbai, India, MH118) and Hugh leifson medium
(HiMedia, M826S). Plates were then incubated for 24–48 h at
37 °C. Two tests were performed for identification of S. aureus,
namely, Gram staining and methyl red-Voges Proskauer (MR-VP)
test [25]. Some biochemical tests such as catalase, coagulase, urease,
citrate, lactose, sucrose and lipid hydrolysis were also performed for
identification of all clinical and nonclinical isolates [26].
2.2. Preparation of leaf extract
Fresh leaves of C. sativa,T. orientalis and P. guajava were col-
lected in January 2016 from the Saaii College of Medical Science
and Technology campus area, Kanpur, Uttar Pradesh, India. For
the purpose of identification, small twigs with reproductive struc-
tures were also collected, dried, mounted on herbarium sheets and
submitted to the Herbarium Facility at North Bengal University,
Siliguri, West Bengal, with the accession numbers 7825 (C. sativa),
7826 (T. orientalis) and 7827 (P. guajava). After collection, leaves
were washed with distilled water, and then dried with blotting
paper in a hot air oven (Spac-N-Service, Kolkata, India) at 60 °C
for 2–3 days. The dried leaves from each plant (50 g) were crushed
separately in 100 mL of ethanol (Merck, 1009831000, Mumbai,
India) at ambient temperature (37–40 °C) and soaked for a period
of 24–48 h. Then the extracts were filtered using Whatman No.1
filter paper (Whatman
TM
, Cat # 1001090, Kent, UK). After that, using
a syringe, extracts were further filtered through 0.22 mm millipore
membrane filters (Merck, SLGV 033RS, Mumbai, India). The result-
ing, active, pigment-free filtrates of the three different leaf extracts
were poured into sterile culture tubes (Riviera, 23175115,
Mumbai, India). Finally, 5 mL of these filtered extracts were kept
aseptically at 4 °C until use. Dilutions of the extracts (10%) were
also prepared during this time [27].
2.3. Phytochemical tests to characterize the active constituents
Some phytochemical tests for bioactive constituents such as
alkaloids, flavonoids, glycosides, phenol, tannins, resins, terpenes
and steroids were carried out on the portion of residual material
using standard phytochemical procedure [28–31].
2.4. Screening of plant extract for antibacterial activity
Antibacterial activities of the plant extracts were determined
using the disc diffusion method of Bauer et al. [32]. Prior to sensi-
tivity testing, each of the 20 experimental bacterial strains was cul-
tured in a petri plate (containing MSA media) and incubated for
18–24 h at 37 °C. A single colony was then cultured in 5 mL
Mueller–Hinton broth (HiMedia, M391) for 4–6 h at 37 °C. Then a
sterile swab was used to inoculate lawn cultures on Mueller–Hinton
agar (MHA) plates (HiMedia, M173). The plates were dried for
15 min and then used for the sensitivity tests. The discs, which
had been loaded with a series of plant extracts, were placed on the
nutrient agar surface. Each test plate had between 1 to 4 experimen-
tal discs. One positive control was prepared with standard commer-
cial antibiotic discs. The standard antibiotic discs used with S. aureus
were penicillin, methicillin, cefoxitin and vancomycin.The plate was
then incubated at 37 °C for 18–24 h, depending on the species of
bacteria used in the test. After the incubation period, the plates were
examined for the inhibition zone around each disc. The diameter of
any inhibition zone was measured with slide calipers (De Tech,
Kolkata, India) and recorded. The tests were repeated three times
to ensure reliability by following National Committee for Clinical
Laboratory Standards guidelines [33,34].
For discs with a single plant extract, 5 mL was loaded on a 5 mm
diameter sterile Whatman filter paper disc and placed on a lawn of
S. Chakraborty et al. / Journal of Integrative Medicine 16 (2018) 350–357 351
S. aureus suspension on a MHA plate. For the synergistic study,
three different combinations of plant extract were used and mixed
in equal amounts. Then 5 mL of the mixture was loaded on a 5 mm
diameter sterile Whatman filter paper disc and kept on a lawn of
S.aureus suspension on MHA plate. The tested combinations used
the ethanolic extracts of C. sativa +T. orientalis,C. sativa +P. guajava
and P. guajava +T. orientalis. In these tests, the zone of inhibition
was recorded after 24 h of incubation.
2.5. High-performance thin-layer chromatography analysis of leave
extracts
The active ethanolic extract was further studied to characterize
its high-performance thin-layer chromatography (HPTLC) finger
print and a phytochemical analysis was conducted [35,36]. HPTLC
was used to detect phytochemical compounds such as quercetin,
gallic acid and catechin, using the ternary mobile phase of
n-butanol:glacial acetic acid:water (80:6:20) on precoated silica
gel plates (20 cm 10 cm, thin-layer chromatography (TLC)
aluminum plates, 105554, Merck, Germany). The TLC plate was
prewashed with methanol and activated at 110 °C for 5 min. The
sample was applied as a 4 mm band, using a Camag Linomat IV
sample applicator (Muttenz, Switzerland), equipped with a
100
l
L syringe. A constant application rate of 5
l
L/s was used.
The identification of quercetin, gallic acid and catechin biomarkers
was conducted and validated with ternary mobile phase n-butanol:
glacial acetic acid:water (80:6:20) on precoated silica gel 60F254
aluminum plates, and densitometric determination was carried
out after derivatization with anisaldehyde-sulfuric acid reagent
in k366-UV absorption-reflectance mode with the standard solu-
tions of the compounds as a reference. The standard solutions were
analyzed at a concentration of 0.1 mg/mL. Chromatograms were
scanned at 350, 290 and 270 nm for quercetin, gallic acid and
catechin, respectively.
The HPTLC method was used for quantification of quercetin,
gallic acid and catechin in different plant extracts. All chemicals,
solvents and reagents were of analytical grade (Merck, Mumbai,
India). Extracts were combined, filtered, and concentrated on a
rotary evaporator (Super fit continental Pvt Ltd, R-150, Mumbai,
India) and further evaporated to dryness in a freeze drier (Eyela
freeze dryer, FDU-506, Tokyo Rikakikai, Tokyo, Japan).
HPTLC-grade methanol (Merck, 106007) was used to dissolve these
freeze-dried extracts during sample preparation [37].
2.6. Statistical analysis
Data in the tables and figures are presented as mean ± standard
error of five independent measurements, unless otherwise stated.
Results were analyzed statistically using a one-way analysis of
variance followed by Duncan’s multiple range test (P< 0.05) in
SPSS version 22 software, wherever appropriate.
3. Results
3.1. Isolation and identification of microbial strains
Ten clinical and 10 nonclinical samples were cultured on MSA
plate and incubated overnight. After incubation, Petri dishes
showed yellow and transparent colonies which were designated
as S. aureus. All the strains showed a positive result in Gram reac-
tion, coagulase test and negative results in MR-VP test. Maximum
strains showed less than 10 mm inhibition zone with 30 mg methi-
cillin disc on MHA medium (Table 1 and Fig. 1).
3.2. Phytochemical tests to characterize the active constituents present
in the leaf extract
Alkaloids, flavonoids, glycosides, resins, terpenes and steroids
were present in the ethanolic extract of C. sativa. Similarly,
alkaloids, flavonoids, saponin, phenols, tannins, terpenes and
steroids were present in both ethanolic extracts of P. guajava and
T. orientalis, but glycosides, resins and steroids were absent
(Table 2).
3.3. Screening of plant extract for antibacterial activity
In the present study, ethanolic plant extracts showed greater
inhibition zones against MRSA strains than common antibiotics
available in the local market. When applied singly, the largest zone
(mean diameter) of inhibition in a nonpatient MRSA culture was
caused by T. orientalis ((29.80 ± 0.71) mm) followed by P. guajava
((29.69 ± 0.78) mm). The largest zone of inhibition (mean
diameter) in an MRSA culture from a patient was caused by
P. guajava ((24.73 ± 0.55) mm). When the plant extracts were used
in combination, larger zones of inhibition were formed against
some strains, clearly indicating the potential for synergistic effect
Table 1
Biochemical characteristics of isolated MRSA strains.
Culture No. Isolate code Coagulase test Catalase test Urease test Citrate test Lactose test Sucrose test Lipid hydrolysis
1 Sh1 +ve +ve +ve +ve +ve +ve +ve
2 Sh2 +ve +ve +ve +ve +ve +ve +ve
3 Sh3 +ve +ve +ve +ve +ve +ve +ve
4 Sh4 +ve +ve +ve +ve +ve +ve +ve
5 Sh5 +ve +ve +ve +ve +ve +ve +ve
6 Sh6 +ve +ve +ve +ve +ve +ve +ve
7 Sh7 +ve +ve +ve +ve +ve +ve +ve
8 Sh8 +ve +ve +ve +ve +ve +ve +ve
9 Sh9 +ve +ve +ve +ve +ve +ve +ve
10 Sh10 +ve +ve +ve +ve +ve +ve +ve
11 G1 +ve +ve +ve +ve +ve +ve +ve
12 G2 +ve +ve +ve +ve +ve +ve +ve
13 G3 +ve +ve +ve +ve +ve +ve +ve
14 G4 +ve +ve +ve +ve +ve +ve +ve
15 G5 +ve +ve +ve +ve +ve +ve +ve
16 G6 +ve +ve +ve +ve +ve +ve +ve
17 G7 +ve +ve +ve +ve +ve +ve +ve
18 G8 +ve +ve +ve +ve +ve +ve +ve
19 G9 +ve +ve +ve +ve +ve +ve +ve
20 G10 +ve +ve +ve +ve +ve +ve +ve
MRSA: methicillin-resistant Staphylococcus aureus; isolate code: strain code; +ve: presence of the character; Sh: nonclinical isolates; G: clinical isolates.
352 S. Chakraborty et al. / Journal of Integrative Medicine 16 (2018) 350–357
of plant extracts. In nonpatient samples, the largest zone of
inhibition was recorded in case of C. sativa +T. orientalis (mean
diameter: (33.59 ± 0.94) mm), followed by C. sativa +P. guajava
(mean diameter: (29.43 ± 0.82) mm) and T. orientalis + P. guajava
(mean diameter: (20.99 ± 0.54) mm). However, greatest
zone against individual strain (Sh1) was recorded by C. sativa +
T. orientalis ((47.41 ± 2.33) mm, Fig. 2). In clinical samples, the
combination of C. sativa +P. orientalis resulted in the greatest zone
of inhibition (mean diameter: (28.77 ± 0.73) mm), followed by
C. sativa +P. guajava ((24.47 ± 0.53) mm) and T. orientalis +
P. guajava ((21.70 ± 0.53) mm)) (Fig. 3). In most of the cases our
experimental plant extracts showed better inhibition of colony
formation than vancomycin did against the MRSA strains used in
the present study (Figs. 2 and 3).
3.4. Detection of phytochemical compounds using HPTLC
The HPTLC analysis confirmed the segregation of a number of
individual compounds in each of the three extracts and yielded
individual retention factor (Rf) values and peak area percentage
or area under the curve (Table 3). The HPTLC finger printing of
P. guajava extract gave 12 spots, C. sativa gave 11 spots, and 13
peaks were observed in case of T. orientalis (Fig. 4).The purity of
the sample extracts was confirmed by comparing the absorption
spectra at the start, middle and end positions of the band of
standard solutions (Figs. 4 and 5).
4. Discussion
Currently, MRSA is a major cause of health care and
community-associated infections worldwide. Nosocomial infec-
tions, acquired by patients from institutional health care, have long
been the typical presentation of MRSA infections. Traditionally, the
risk factors for MRSA infection have included hospital care, chronic
care facilities and nursing homes for elderly people, the presence of
indwelling devices or chronic wounds, and previous antibiotic
treatment. MRSA frequently causes illness in people with a com-
promised immune system who interact with or reside in hospitals
and health care facilities. Health care-associated MRSA or
‘‘HA-MRSA” is often acquired through a wound and any other
Fig. 1. Staphylococcus aureus on mannitol salt agar plate.
Table 2
Phytochemical analysis of ethanolic plant leaf extracts.
Phytochemical Color Cannabis
sativa
Thuja
orientalis
Psidium
guajava
Alkaloids Orange +++
Saponin ++
Flavonoids Light yellow +++
Tannins ++
Cardiac
glycosides
Reddish
brown
++
Phenols Bluish black +++
Terpenes Reddish
brown
+++
Resins Violet ++
Steroids Brown ++
+: positive; : negative.
Fig. 2. Antibacterial activity of standard antibiotics and plant extracts against methicillin-resistant nonclinical isolates. Sh: nonclinical isolates. For each strain, different letter
above each column indicates significant difference between the zones of inhibition with P< 0.05 based on Duncan’s multiple range test.
S. Chakraborty et al. / Journal of Integrative Medicine 16 (2018) 350–357 353
Fig. 3. Anti-methicillin-resistant Staphylococcus aureus activity of common antibiotics and ethanolic leaf extracts against clinical isolates. G: clinical isolates. For each strain,
different letter above each column indicates significant difference between the zones of inhibition with P< 0.05 based on Duncan’s multiple range test for each strain.
Table 3
Phytochemical analysis of ethanolic leaf extracts for the presence of quercetin, gallic acid and catechin by HPTLC method.
Serial No. Sample ID Sample name Loading volume (mL) AUC (mg/(mLmin) Wavelength (nm)
1 Standard 1 Quercetin 1 1 16,168.9 350
2 Standard 2 Quercetin 2 2 32,772.8
3 Standard 3 Quercetin 3 3 40,186.1
4 Standard 4 Quercetin 4 4 41,007.1
5 Sample 5 Psidium guajava 8 1,851.2
6 Sample 6 Cannabis sativa 8 2,093.8
7 Sample 7 Thuja orientalis 8 4,421.6
8 Standard 1 Gallic acid 1 1 8,305.7 290
9 Standard 2 Gallic acid 2 2 8,962.8
10 Standard 3 Gallic acid 3 3 12,767.5
11 Standard 4 Gallic acid 4 4 25,154.8
12 Sample 5 Psidium guajava 8 2,741.3
13 Sample 6 Cannabis sativa 8 Not detected
14 Sample 7 Thuja orientalis 8 Not detected
15 Standard 1 Catechin 1 1 8,864.5 270
16 Standard 2 Catechin 2 2 10,217.5
17 Standard 3 Catechin 3 3 11,665.3
18 Standard 4 Catechin 4 4 27,576.3
19 Sample 5 Psidium guajava 8 3,652.2
20 Sample 6 Cannabis sativa 8 Not detected
21 Sample 7 Thuja orientalis 8 Not detected
No.: number; ID: identity; AUC: the area under curve; HPTLC: high-performance thin-layer chromatography.
Fig. 4. High-performance thin-layer chromatography chromatogram of Psidium guajava (A), Cannabis sativa (B) and Thuja orientalis (C) leaf extract. AU: absorption unit; Rf:
retention factor.
354 S. Chakraborty et al. / Journal of Integrative Medicine 16 (2018) 350–357
opening of skin; other risk factors include old age-related multiple
complex health issues, weakened immune systems, prolonged
dialysis and cancer treatment, resulting in a compromised immune
system.
Many different definitions for multidrug-resistant (MDR), exten-
sively drug-resistant (XDR) and pan-drug-resistant (PDR) bacteria
are being used in the medical literature to characterize the different
patterns of resistance found in HA-MASA. Magiorakos et al. [38]
described the acquired resistance profiles of S. aureus,Enterococcus
spp., Enterobacteriaceae (other than Salmonella and Shigella), Pseu-
domonas aeruginosa and Acinetobacter spp. These bacteria are often
responsible for health care-associated infections and are known to
have strains with MDR. Currently, the problem of antimicrobial
resistance in bacteria is prompting the scientific community to
investigate MDR, XDR and PDR throughout the world [38].
In the present study, we have evaluated the efficiency of
ethanolic extracts of C. sativa,T. orientalis and P. guajava against
MRSA. A total of 20 S. aureus strains were isolated from clinical
and nonclinical settings. All coagulase-positive MRSA isolates were
chosen for inhibitory studies if they had a zone of inhibition smal-
ler than 10 mm in response to a 30 mg methicillin disc on MHA. All
20 isolates showed positive results for catalase, coagulase, urease,
citrate, lactose and sucrose tests and lipid hydrolysis. Coagulase-
positive MRSA isolates are commonly antibiotic resistant [39,40].
The treatment of S. aureus infections is becoming increasingly
more complicated and tough, due to the emergence of various
types of antibiotic resistance [41]. We performed an antibiotic sen-
sitivity test with the help of known antibiotics that are commonly
available in the market and mostly used against MRSA infections,
including penicillin, methicillin, oxacillin, cefoxitin and van-
comycin. Our results showed that most of the S. aureus strains were
resistant to penicillin, methicillin, oxacillin and cefoxitin; some of
the strains were still susceptible to vancomycin.
In recent years, there have been ever increasing reports about
the anti-microbial activity of plant, extracts. MRSA, which is
known to be resistant to methicillin and other b-lactam antibiotics,
has been found to be vulnerable to inhibition by various plant
extracts. Alkaloids, flavonoids, cardiac glycosides, reducing sugars,
saponins, resins, terpenes, terpenoids and steroids are principle
phytochemical compounds present in plants. In the present study,
MDR, lactamase-producing MRSA strains were used to determine
the antibacterial activity of three Indian medicinal plants C. sativa,
T. orientalis and P. guajava, alone and in combination. From the
available literature, the major phytochemicals present in P. guajava
leaves include flavonoids, tannins, triterpenoids, saponins, sterols,
phenols, glycosides, alkaloids and carbohydrates [42–46]. Phyto-
chemicals are chemical compounds synthesized during the various
metabolic processes, and may have a variety of pharmacological
activities in animals. Some of these are found to have antimicrobial
activity against pathogenic organisms and can serve crucial roles in
plant defense mechanisms. These compounds are mainly classified
as phenols, quinones, flavonoids, tannins, alkaloids, glycosides and
polysaccharides.
In this experimental work, we found that alkaloids, saponins,
flavonoids, steroids, cardiac glycosides, terpenes, resins, tannins
and phenols were present in P. guajava and C. sativa, while sapo-
nins and tannins were absent; steroids, resins, and cardiac glyco-
sides were also absent in T. orientalis. When used in isolation
against MRSA, the zone of inhibition created by ethanolic extracts of
these three Indian medicinal plants spanned the range of 10–48 mm;
when applied in combination, better results were observed. Phyto-
chemical analysis using HPTLC revealed the presence of alkaloids,
saponins, flavonoids, phenols and tannins in the ethanolic leaf
extracts of T. orientalis,P. guajava and C. sativa. HPTLC finger print-
ing studies confirmed the results of phytochemical screening
through the presence of various bands visualized at different wave
lengths and with specific solvent systems, showing the presence of
particular phytochemical compounds [47]. We found catechin as a
flavonoid and gallic acid as a phenolic; both compounds were pre-
sent in P. guajava. Phenolic compounds such as quercetin were pre-
sent in all three tested ethanolic plant extracts. There was a
profound synergism between C. sativa and T. orientalis (1:1), and
between P. guajava and C. sativa (1:1), as shown by their larger
zones of inhibition. This synergy may be due to the presence of
phenolics (e.g., quercetin and gallic acid) and flavonoids (e.g.,
catechin) in the combined leaf extracts. The detailed interaction
will be the topic of a future investigation by our group. Gallic acid
has a potential antimicrobial activity against Campylobacter, but its
effectiveness has been poorly studied. Treatment with gallic acid
resulted in a loss of calcium ions from susceptible strains ((0.58–
1.53)
l
g/mL and (0.54–1.17)
l
g/mL, respectively, over a 180 min
period) but not in the controls [48]. The quercetin has antimicro-
bial effects that are at least partially attributed to inhibition of
deoxyribonucleic acid gyrase [49]. Similarly, catechin apparently
has selective bactericidal activity against pathogenic bacteria
[50]. When we used ethanolic leaf extracts for analysis of synergis-
tic effects, we observed maximum zones of inhibition against tested
pathogenic strains due to the presence of some phytochemical com-
pounds in those leaf extracts. Some phytochemical compounds, like
steroids, anthraquinones, resins, volatile oils, terpenes, steroids, car-
diac glycosides and phlobatannins, did not show any antimicrobial
activity against S. aureus,B. subtilis,E. coli,P. aeruginosa and Sal-
monella sp. [51]. Many researchers have reported a synergistic effect
of antibiotics and plant-based therapeutics. In our study, all these
plant extracts showed promising antibacterial activity against all
the clinical and nonclinical test strains.
Fig. 5. Standard chromatogram of catechin (A), gallic acid (B) and quercetin (C). AU: absorption unit; Rf: retention factor.
S. Chakraborty et al. / Journal of Integrative Medicine 16 (2018) 350–357 355
5. Conclusions
In conclusion, the selected strains of MRSA were found to be
sensitive to the slightly higher concentration of crude ethanol
extracts. Ethanolic extract of C. sativa alone and in combination
with T. orientalis provided two potential therapeutic agents for
use against MRSA infections. However, a toxicity test is still needed
to be done to confirm safety of these extracts. These extracts may
also be useful for developing a formulation for topical use in der-
mal infection like sepsis, acne, pimples and carbuncles.
Acknowledgements
The authors are grateful to Saaii Educational Foundation,
Kanpur-209203, India, for their financial assistance. The authors
are also thankful to Dr. Manoranjan Chowdhury of North Bengal
University for proper identification of the plants.
Competing interests
The authors declare that they have no competing interests.
References
[1] Deurenberg RH, Vink C, Kalenic S, Friedrich AW, Bruggeman CA, Stobberingh
EE. The molecular evolution of methicillin resistant Staphylococcus aureus. Clin
Microbiol Infect 2007;13(3):222–35.
[2] Rolinson GN. ‘‘Celbenin”-resistant Staphylococci. Br Med J 1961;1(5219):125–6.
[3] Rasmussen G, Monecke S, Ehricht R, Söderquist B. Prevalence of clonal
complexes and virulence genes among commensal and invasive Staphylococcus
aureus isolates in Sweden. PLoS One 2013;8(10):e77477.
[4] Ghirardi B, Pietrasanta C, Ciuffini F, Manca MF, Uccella S, Lavizzari A, et al.
Management of out breaks of nosocomial pathogens in neonatal intensive care
unit. Pediatr Med Chir 2013;35(6):263–8.
[5] Xiao M, Wang H, Zhao Y, Mao LL, Brown M, Yu YS, et al. National surveillance of
methicillin-resistant Staphylococcus aureus in China highlights a still-evolving
epidemiology with 15 novel emerging multilocus sequence types. J Clin
Microbiol 2013;51(11):3638–44.
[6] Nemati M, Hermans K, Lipinska U, Denis O, Deplano A, Struelens M, et al.
Antimicrobial resistance of old and recent Staphylococcus aureus isolates from
poultry: first detection of livestock-associated methicillin-resistant strain
ST398. Antimicrob Agents Chemother 2008;52(10):3817–9.
[7] Moore CL, Hingwe A, Donabedian SM, Perri MB, Davis SL, Haque NZ, et al.
Comparative evaluation of epidemiology and outcomes of methicillin-resistant
Staphylococcus aureus (MRSA) USA300 infections causing community- and
healthcare-associated infections. Int J Antimicrob Agents 2009;34(2):148–55.
[8] Skov RL, Jensen KS. Community-associated methicillin-resistant Staphylococcus
aureus as a cause of hospital-acquired infections. J Hosp Infect 2009;73
(4):364–70.
[9] van Rijen MM, van Keulen PH, Kluytmans JA. Increase in a Dutch hospital of
methicillin-resistant Staphylococcus aureus related to animal farming. Clin
Infect Dis 2008;46(2):261–3.
[10] Eveillard M, Martin Y, Hidri N, Boussougant Y, Joly-Guillou ML. Carriage of
methicillin-resistant Staphylococcus aureus among hospital employees:
prevalence, duration, and transmission to households. Infect Control Hosp
Epidemiol 2004;25(2):114–20.
[11] Bywater R, Deluyker H, Deroover E, de Jong A, Marion H, McConville M, et al. A
European survey of antimicrobial susceptibility among zoonotic and
commensal bacteria isolated from food-producing animals. J Antimicrob
Chemother 2004;54(4):744–54.
[12] de Boer E, Zwartkruis-Nahuis JT, Wit B, Huijsdens XW, de Neeling AJ, Bosch T,
et al. Prevalence of methicillin-resistant Staphylococcus aureus in meat. Intl J
Food Microbiol 2009;134(1–2):52–6.
[13] Normanno G, Corrente M, LaSalandra G, Dambrosio A, Quaglia NC, Parisi A,
et al. Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal
origin product in Italy. Int J Food Microbiol 2007;117(2):219–22.
[14] Li L, Ye L, Yu L, Zhou C, Meng H. Characterization of extended spectrum b-
lactamase producing enterobacteria and methicillin-resistant Staphylococcus
aureus isolated from raw pork and cooked pork products in south China. J Food
Sci 2016;81(7):1773–7.
[15] Costa WLR, Ferreira JDS, Carvalho JS, Cerqueira ES, Oliveira LC, CastroAlmeida
RCD. Methicillin-resistant Staphylococcus aureus in raw meats and prepared
foods in public hospitals in Salvador, Bahia. Brazil. J Food Sci 2015;80
(1):147–50.
[16] Adwan G, Mhanna M. Synergistic effects of plant extracts and antibiotics on
Staphylococcus aureus strains isolated from clinical specimens. Middle East J
Sci Res 2008;3(3):134–9.
[17] Oluduro AO, Omoboye O. In vitro antibacterial potentials and synergistic effect
of south-western Nigerian plant parts used in folklore remedy for Salmonella
typhi infection. Nat Sci 2010;8(9):52–9.
[18] Gertsch J. Botanical drugs, synergy, and network pharmacology: forth and back
to intelligent mixtures. Planta Med 2011;77(11):1086–98.
[19] György É. Study of the antimicrobial activity and synergistic effect of some
plant extracts and essential oils. Rev Româna
˘Med Lab 2010;18:49–56.
[20] Moussaoui F, Alaoui T. Evaluation of antibacterial activity and synergistic
effect between antibiotic and the essential oils of some medicinal plants. Asian
Pac J Trop Biomed 2016;6(1):32–7.
[21] Chanda S, Rakholiya KD. Combination therapy: synergism between natural
plant extracts and antibiotics against infectious diseases. Sci Microb Pathog
2011:520–9.
[22] Rakholiya KD, Kaneria MJ, Chanda SV. Medicinal plants as alternative sources
of therapeutics against multidrug-resistant pathogenic microorganisms based
on their antimicrobial potential and synergistic properties. In: Rai MK, Kon KV,
editors. Fighting multidrug resistance with herbal extracts, essential oils and
their components. Oxford: Elsevier Inc; 2013. p. 165–79.
[23] Chovanová R, Mikulášová M, Vaverková Š. In vitro antibacterial and antibiotic
resistance modifying effect of bioactive plant extracts on methicillin-resistant
Staphylococcus epidermidis. Int J Microb 2013;2013:1–7.
[24] Blesson J, Saji CV, Nivya RM, Kumar R. Synergism of antibiotics and plant
extracts in antibacterial activity against methicillin resistant Staphylococcus
aureus (MRSA). World J Pharm Pharm Sci 2014;4(3):741–63.
[25] Baird-Parker AC. A classification of micrococci and staphylococci based on
physiological and biochemical tests. Microbiology 1963;30(3):409–27.
[26] Addis M, Pal M, Kyule MN. Isolation and identification of Staphylococcus
species from raw bovine milk in Debre Zeit, Ethiopia. Vet Res 2011;4
(1):13–7.
[27] Mailoa MN, Laga A, Djide N. Tannin extract of guava leaves (Psidium guajava L)
variation with concentration organic solvents. Int J Sci Technol Res 2013;2
(9):106–10.
[28] Helrich K. Association of official analytical chemists (AOAC). In: Helrich K.
Official method of analysis, 15th ed., Arlington, Virginia; 1990.
[29] Joseph B, Priya RM. Review on nutritional medicinal and pharmacological
properties of Psidium guajava. Int J Pharma Bio Sci 2011;124(2):53–69.
[30] AtesßDA, Turgay Ö. Antimicrobial activities of various medicinal and
commercial plant extracts. Turk J Biol 2003;27:157–62.
[31] Jasuja ND, Sharma SK, Saxena R, Choudhary J, Sharma R, Joshi SC. Antibacterial,
antioxidant and phytochemical investigation of Thuja orientalis leaves. J Med
Plants Res 2013;7(25):1886–93.
[32] Bauer AW, Kirby E, Sherris EM, Turk M. Antibiotic by standardized single disk
method. Am J Clin Path 1996;45(4):493–6.
[33] NCCLS Reference method for broth dilution antifungal susceptibility testing of
yeasts; approved standard—second edition 2002 Wayne, Pennsylvania: NCCLS.
[34] Clinical and Laboratory Standards Institute. Reference method for broth
dilution antifungal susceptibility testing filamentous fungi, approved
standard—second edition. Wayne, Pennsylvania: Clinical and Laboratory
Standards Institute; 2008.
[35] Kalaivanan M, Jesudoss LL, Ganthi AS, Subramanian MPS. Preliminary
phytochemical screening and HPTLC finger print profile of Tragia plukenetii.
Int J Pharm Sci Res 2016;8(3):1194–8.
[36] Arokiyaraj S, Perinbam K, Agastian P, Kumar RM. Phytochemical analysis and
antibacterial activity of Vitex agnus-castus. Int J Green Pharm 2009;3
(2):162–4.
[37] Reddy BS, Reddy RKK, Naidu VGM, Madhusudhana K, Agwane SB, Ramakrishna
S, et al. Evaluation of antimicrobial, antioxidant and wound healing potentials
of Holoptelea integrifolia. J Ethnopharmacol 2008;115(2):249–56.
[38] Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al.
Multidrug-resistant, extensively drug-resistant and pandrug-resistant
bacteria: an international expert proposal for interim standard definitions
for acquired resistance. Clin Microbiol Infect 2012;18(3):268–81.
[39] Woodford N, Livermore DM. Infections caused by Gram-positive bacteria: a
review of the global challenge. J Infect 2009;59(Suppl. 1). S4–16.
[40] El-Hadedy D, El-Nour SA. Identification of Staphylococcus aureus and
Escherichia coli isolated from Egyptian food by conventional and molecular
methods. J Genetic Eng Biotech 2012;10(1):129–35.
[41] Dhanalakshmi TA, Umapathy BL, Mohan DR. Prevalence of methicillin,
vancomycin and multidrug resistance among Staphylococcus aureus. J Clin
Diagn Res 2012;6(6):974–7.
[42] Chopra RN, Nayer SL, Chopra IC. Glossary of Indian medicinal plants. 3rd
ed. New Delhi: CSIR; 1992. p. 7–246.
[43] Arya V, Thakur N, Kashyap CP. Preliminary phytochemical analysis of the
extracts of Psidium leaves. J Pharmacog Phytochem 2012;1(1):1–5.
[44] Biswas B, Rogers K, McLaughlin F, Daniels D, Yadav A. Antimicrobial activities
of leaf extracts of guava (Psidium guajava L.) on two Gram-negative and Gram-
positive bacteria. Int J Microbiol 2016;2(6):375–8.
[45] Khubeiz MJ, Mansour G, Zahraa B. Antibacterial and phytochemical
investigation of Thuja orientalis (L.) leaves essential oil from Syria. Int J
Current Pharm Rev Res 2016;7(5):243–7.
[46] Jasuja ND, Sharma SK, Saxena R, Chaudhary J, Sharma R. Antibacterial,
antioxidant and phytochemical investigation of Thuja orientalis leaves. J Med
Plants Res 2013;7(25):1886–93.
[47] Seasotiya L, Siwach P, Malik A, Bai S, Bharti P, Dalal S. Phytochemical
evaluation and HPTLC finger print profile of Cassia fistula. Int J Adv Pharm Biol
Chem 2014;3(3):604–11.
356 S. Chakraborty et al. / Journal of Integrative Medicine 16 (2018) 350–357
[48] Sarjit A, Wang Y, Dykes GA. Antimicrobial activity of gallic acid against
thermophilic Campylobacter is strain specific and associated with a loss of
calcium ions. Food Microbiol 2015;46:227–33.
[49] Cushnie TPT, Lamb AJ. Antimicrobial activity of flavonoids. Int J Antimicrob
Agents 2005;26(5):343–56.
[50] Hara Y. Food phytochemicals for cancer prevention II. Washington,
DC: American Chemical Society; 1994. p. 34–50.
[51] Kunle OF, Egharevba HO, Ibrahim J, Iliya I, Abdullahi MS, Okwute SK.
Antimicrobial activity of the extract of Vernonia ambigua (aerial part).
Researcher 2010;2(6):74–80.
S. Chakraborty et al. / Journal of Integrative Medicine 16 (2018) 350–357 357
