Journal of King Saud University – Science (2017) 29, 84–95

King Saud University

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ORIGINAL ARTICLE

In vitro antibacterial activity of crude extracts of 9

selected medicinal plants against UTI causing
MDR bacteria
Monali P. Mishra a, Sibanarayan Rath a, Shasank S. Swain a, Goutam Ghosh b,
Debajyoti Das b, Rabindra N. Padhy a,*

a
Central Research Laboratory, Institute of Medical Sciences and Sum Hospital, Siksha ‘O’ Anusandhan University, Bhubaneswar
751003, Odisha, India
b
School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan University, Bhubaneswar 751003, Odisha, India

Received 13 October 2014; accepted 24 May 2015

Available online 28 May 2015

KEYWORDS Abstract Urinary tract infection (UTI) has become a more grievous problem today, due to mul-
Tropical plants; tidrug resistance of infecting Gram-positive (GP) and Gram-negative (GN) bacteria, sometimes
Urinary tract infection; even with multiple infections. This study examines effectivity of 9 tropical flowering plants
Complementary medicine; (Anogeissus acuminata, Azadirachta indica, Bauhinia variegata, Boerhaavia diffusa, Punica grana-
Multidrug resistant bacteria; tum, Soymida febrifuga, Terminalia chebula, Tinospora cordifolia and Tribulus terrestris) for possible
In vitro antibacterial activity use as source of antimicrobials for multidrug resistant (MDR) bacteria, along with main-stream
antibiotics. Pathogenic bacteria were isolated from urine samples of patients attending and admit-
ted in the hospital. Antibiograms of 11 isolated bacteria (GPs, Enterococcus faecalis and
Staphylococcus aureus; and GNs, Acinetobacter baumannii, Citrobacter freundii, Enterobacter aero-
genes, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris
and Pseudomonas aeruginosa) were ascertained by the disc-diffusion method, and antibacterial
effectivity of plant extracts was monitored by the agar-well diffusion method. Isolated bacteria were
floridly MDR to most antibiotics of the day. Methanol extracts of 9 plants were used, and extracts
of 3 plants, A. acuminata, P. granatum and S. febrifuga at least caused 25–29 mm as the maximum
size of zone of inhibition on bacterial lawns. Minimum inhibitory concentration (MIC) and mini-
mum bactericidal concentration (MBC) values of methanol extracts of 9 plants were recorded. The
methanol extract of A. acuminata had 0.29 mg/ml as the lowest MIC value and 0.67 mg/ml as the
lowest MBC value, against MDR S. aureus, signifying effectivity; but, it had the highest MIC value

* Corresponding author. Tel.: +91 9437134982; fax: +91

6742432034.
E-mail address: rnpadhy54@gmail.com (R.N. Padhy).
Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

http://dx.doi.org/10.1016/j.jksus.2015.05.007
1018-3647 ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Antibacterial activity of medicinal plants against MDR bacteria 85

of 3.41 mg/ml. and the highest MBC value of 4.27 mg/ml for most other MDR bacteria including
E. coli. Qualitative phytochemical analysis was done for these 9 plants and information on leading
phytochemicals was presented retrieved from PubChem database. Thus, three effective-most plants
in controlling MDR-UTI bacteria in vitro were A. acuminata, P. granatum and S. febrifuga, which
can be promoted as complementary medicine.
ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction mainstream drugs, the antibiotics, as it takes 3–4 days of time

of arrival of the culture report of the urine during which per-
Urinary tract infection (UTI) was mostly caused by Gram neg- iod, infecting bacteria cause further problems. UTI episodes
ative (GN) bacteria, predominately by Escherichia coli and by need to be controlled with an iron hand. And to avoid the
mixed infections of (Gram positive, GP) Staphylococcus use of any antibiotic of higher generation during empiric ther-
aureus, Enterococcus faecalis, including other GNs, Klebsiella apy, complementary or synergistic therapy using phyto-drugs
pneumoniae, Pseudomonas aeruginosa, Acinetobacter bauman- with any ongoing antibiotic could be prudently used against
nii, Enterobacter aerogenes, Proteus mirabilis, Citrobacter UTI.
freundii, Proteus vulgaris and Klebsiella oxytoca in the decreas- In continuation to previous work on scientific verification of
ing order of prevalence, when monitored in the Institute of ethnomedicinal information of a group of plants from
Medical Sciences and Sum Hospital, for example (Mishra Kalahandi (Odisha) forest (Mishra and Padhy, 2013; Rath
et al., 2013). The infecting bacteria normally constitute the fae- and Padhy, 2014), the cited 9 plants were selected. These plants
cal flora, and the UTI episode is initiated, when the urine flow were too recorded in Indian pharmacopeia (Anonymous, 2014),
in an individual is obstructed by one of several reasons such as, as plants frequently used against general infectious diseases:
strictures, calculi, tumours, prostatic hypertrophy, vesi- A. acuminata is in use for UTI and skin diseases; whole plant
courethral reflux, diabetes, anal disease, pregnancy, catheteri- of A. indica is used as an antiseptic; B. variegata is used against
zation, some surgical procedure at the urinogenital region diarrhoea and throat infections; B. diffusa is used for UTI and
and cystoscopy (Saint et al., 2002). The infecting bacteria dysentery; P. granatum is used for treating diarrhoea, dysentery
invade urethra and bladder with a compromised body defense and throat problems; S. febrifuga is used against diarrhoea,
mechanism and decreased urine flow. In these conditions, the dysentery and UTI; T. chebula is used for diarrhoea and dysen-
bacteria ascend via urethra move to the bladder mucosa, colo- tery; T. cordifolia has anti-tubercular activity; and T. terrestris is
nize, multiply and cause inflammation; this causes intolerable used against UTI. Several other plants, Terminalia alata,
pain, burning, frequency and urgency of urination, nocturia, Lantana camara, Combretum albidum and Woodfordia fruticosa
foul smelling, cloudy urine and haematuria. Indeed, at the had comparable antibacterial activities, which had been used for
onset of the problem, the patient reports to the physician further pharmacognostical studies (Rath and Padhy, 2012; Rath
and an empiric therapy is started before the culture report of and Padhy, 2013; Dubey et al., 2014; Sahu et al., 2014), but these
the urine sample is obtained. 9 cited plants were not used for the isolation of individual com-
If any infection in a patient is not controlled, infecting pounds against any bacteria. Detailed antibacterial work with
microbes get resistance to the applied antibiotics intrinsically pathogenic bacteria isolated from clinical samples of patients
and a drug resistant cell survives and predominates with con- in the hospital, with 9 plants is described.
comitant bacterial genetic exchanges mechanisms (McMurry
and Levy, 2011). In short, there are several factors of antibiotic
resistance in pathogenic bacteria and this situation has become 2. Materials and methods
a clinical consternation. A physician often prescribes some
higher generation antibiotics in empiric therapy to avoid the 2.1. Collection of plants and extract preparation
debacle from treatment failure arising from the possible pres-
ence of MDR bacteria. And the UTI being a graver problem
Plants reported were collected from the Kandha tribe at hills of
than imagined because of frequent attacks in females mainly,
Eastern range of mountains of India, in the district Kalahandi,
some complementary/adjuvant/synergistic therapeutic strategy
Odisha in January 2014. About 50 respondents of 25 hamlets
is warranted.
were interviewed in a forest patch and the recorded informa-
Furthermore, information from ethnobotany/traditional
tion was documented (Table 1, Fig. 1), with the snowball
medicine has been seen useful for several health complications
method of survey and sampling. Methanol extracts of dried
other than infectious diseases. Nine flowering plants
leaf samples dissolved in 10% v/v dimethyl sulfoxide
(Anogeissus acuminata, Azadirachta indica, Bauhinia variegata,
(DMSO) were used, as detailed (Mishra and Padhy, 2013).
Boerhaavia diffusa, Punica granatum, Soymida febrifuga,
Terminalia chebula, Tinospora cordifolia and Tribulus
2.2. Isolation, identification of bacterial strains and antibiotic
terrestris) (Fig. 1) were used. These plants are in use tradition-
sensitivity test
ally by local ethnic tribes against infectious diseases; and these
were examined in a systematic screening with UTI causing bac-
teria for use as source of non-microbial antimicrobials, so that Two GPs, E. faecalis and S. aureus including 9 GNs, A. bau-
these could serve as complementary medicine, along with mannii, C. freundii, E. aerogenes, E. coli, K. oxytoca,
86 M.P. Mishra et al.

Figure 1 Photographs of plants: a, Anogeissus acuminata; b, Azadirachta indica; c, Bauhinia variegata; d, Boerhaavia diffusa; e, Punica
granatum; f, Soymida febrifuga; g, Terminalia chebula; h, Tinospora cordifolia; i, Tribulus terrestris.

K. pneumoniae, P. mirabilis, P. vulgaris and P. aeruginosa were 2.4. Determinations of MIC and MBC of plant extracts
used in this study. All these bacteria were directly isolated from
urine samples of UTI patients attending and other patients Original stock solutions of leaf extracts were prepared with
admitted in the hospital (see, Mishra et al., 2013). methanol, at 44.44 mg plant extract/ml 10% DMSO solution,
Identification of pathogenic bacterial strains was done depend- with distilled water. Each stock solution was diluted to obtain
ing upon gross colony morphology and biochemical tests of final concentrations of 0.29, 0.67, 1.51, 3.41, 4.27, 9.63, 21.67
isolated pure bacterial cultures, along with Microbial Type and 44.44 mg/ml with the DMSO solution. Minimum inhibi-
Culture Collection (MTCC), Chandigarh, reference strains tory concentration (MIC) and minimum bactericidal concen-
(Mishra and Padhy, 2013). All bacterial strains were subjected tration (MBC) of the methanol leaf extracts were determined
to antibiotic sensitivity tests by the Kirby-Bauer’s disc diffu- in a well on a 96-welled (12 · 8) micro-titre plate (Fig. 2), as
sion method, using a 4 mm thick Mueller–Hinton (MH) agar described elsewhere (Mishra and Padhy, 2013; Rath and
(HiMedia, Mumbai) medium, following the standard antibi- Padhy, 2014).
otic susceptibility test chart of Clinical Laboratory Standard
Institute guidelines (CLSI, 2011). The use of urine samples
does warrant ethical approval of the institute. 2.5. Qualitative phytochemical analyses

2.3. Antibacterial test of plant extracts The following tests were performed for selected 9 medicinal
plants for the presence of alkaloids, carbohydrates, saponins,
One strain from each bacterial species having resistance to a flavonoids, steroids/terpenes, tannins, resins, glycosides, and
maximum number of presently used antibiotics was further anthraquinones, as detailed previously (Dubey and Padhy,
used for monitoring antibacterial potentiality of methanol leaf 2013).
extracts with gentamicin 30 lg/ml as the reference standard, by
the agar-well diffusion method, as previously detailed (Mishra 2.6. Statistical analysis
and Padhy, 2013; Rath and Padhy, 2014). Antibacterial activ-
ities were evaluated as before (Mishra and Padhy, 2013) and Kruskal–Wallis H test for data of zone of inhibition as
results of the third repetition are presented. antibacterial activities in agar-cup method of 3 comparable
Antibacterial activity of medicinal plants against MDR bacteria 87

Table 1 Ethnomedicinal report of 9 medicinal plants used against urinary tract infection causing bacteria.
Sl. Plant name Family English name; Parts Ethnomedicinal uses
No Local name used
1 Anogeissus acuminata Combretaceae Button tree; Leaf/ Its leaf has wound healing activity; used for inflammation,
(Roxb.ex.DC) wall.ex Guill. Phasi Bark urinary tract infection (UTI) and skin diseases. Its bark is used to
and perr.) treat diabetes
2 Azadirachta indica L. Adelb Meliaceae Neem; Leaf It is used as an antiseptic and for antiviral action (chicken pox).
Neemba It is used for the treatment of acne
3 Bauhinia variegata L. Fabaceae Mountain Leaf/ Its leaf is used for burning sensation during urination. The roots
ebony; Root are used for digestive problems, diarrhoea and throat infections
Kanchan
4 Boerhaavia diffusa L. nom. Nyctaginaceae Hog weed; Leaf It is used to improve eyesight, to treat UTI, dysentery and
cons. Atika podi diabetes
5 Punica granatum L. Lythraceae Pomegranate; Leaf/ It is used for diarrhoea, dysentery, intestinal parasites, kidney
Dalimba Bark/ problems, heart and throat problems; it is used to stop nose
Fruits bleeds and gum bleeds and as an eye drop to slow the
development of cataracts
6 Soymida febrifuga Roxb. Meliaceae Indian Leaf/ It is used in the treatment of diarrhoea, dysentery, UTI, fever,
redwood; Bark vaginal infections, rheumatism swellings
Rohini
7 Terminalia chebula Retz. Combretaceae Chebulic Leaf/ Its leaves are used in skin disorders, anaemia, narcosis, piles,
myrobala; Fruits fever, diarrhoea, dysentery, cough and UTI; fruits are used for
Harida constipation and anorexia
8 Tinospora cordifolia Menispermaceae Heart-leaved Leaf/ It has hepato-protective activity; commonly it is used for diabetes
(Thunb.) Miers moonseed; Bark and also to treat tuberculosis
Guluchi
9 Tribulus terrestris L. Zygophyllaceae Puncture vine; Leaf It is used to treat kidney, bladder, UTI and sexual problems
Gokhura

3. Results

3.1. Collection of plants

Ethnomedicinal information on 9 selected medicinal plants are

documented along with the details of modalities on crude
extracts as medicine for many ailments used by local ethnic
aborigine groups (Table 1). Most of these plants are used for
infectious diseases and were found edible as medicines by the
aborigine society.

3.2. Antibiotic sensitivity test of bacteria

The antibiotic profile of each pathogenic bacterium was deter-

mined using specified antibiotic discs (Table 2). GP isolates, E.
faecalis were resistant to 17 and S. aureus were resistant to 13
of 18 antibiotics used. Among the nine GN isolates, A. bau-
Figure 2 Determination of minimum inhibitory concentrations mannii, E. aerogenes, and E. coli were resistant to 11, C. fre-
(MICs), in a 96-well microtiter plate, of 44.44 mg/ml of methanol undii, K. pneumoniae, K. oxytoca and P. aeruginosa were
leaf extract of P. granatum against 8 MDR UTI causing resistant to 12, P. mirabilis and P. vulgaris were resistant to
pathogenic bacteria (1 = S. aureus, 2 = E. faecalis, 3 = 10 antibiotics of the total 14 antibiotics used. The details of
A. baumannii, 4 = E. aerogenes, 5 = K. pneumoniae, 6 = K. individual antibiotics resistant profiles of individual bacteria
oxytoca, 7 = P. mirabilis, 8 = P. vulgaris). M = MIC at numbers are presented (Table 2). Thus, all isolated bacterial strains were
that signifies the lowest concentration of leaf extract. C = control MDR.
without plant leaf extract; A = Gentamicin (30 lg/ml) as control
without any plant leaf extract. 3.3. Antibacterial test of plant extracts

Methanol extracts of medicinal plants when tested against

plants against 11 bacteria was done using the Statistical MDR strains of 11 bacteria, 3 plants, A. acuminata, P. grana-
Package for Medical Science version 17.0 (SPSS Inc., IL, tum and S. febrifuga were seen most effective, with at least
USA). causing 25 to 29 mm diameter-sizes of zone of inhibition
88 M.P. Mishra et al.

Table 2 Antibiotic susceptibility results of multidrug resistant Gram-positive and Gram-negative bacteria.
Bacterium Susceptibility to prescribed antibiotics
Aminoglycosides b-lactams Cephalosporin Fluoroquinolones Glyco- Lincosa-mide Sulfonamide Stand
peptides alones
Ac Ge Ak Am Ox Pt Ce Cf Of Le Nx Gt Tei Va Cd Cot Ch Lz
*
E. faecalis R R R R R R R R R R R R R S R R R R
S. aureus* R R R R MS R R R R R R R MS MS MS R R S
A. baumannii R R R R ND R R R R R MS S ND ND ND R R S
C. freundii R R R R ND R R R R R R MS ND ND ND R R S
E. aerogenes R R R R ND R R R R R R MS ND ND ND R MS S
E. coli R R R R ND S R R R R R R ND ND ND S R S
K. oxytoca R R R R ND R R R R R R MS ND ND ND S R R
K. pneumoniae R R R R ND R R R R R R S ND ND ND R R S
P. mirabilis R R R R ND S R R S R S MS ND ND ND R R R
P. vulgaris R R R S ND R R S S R R S ND ND ND R R R
P. aeruginosa R R R R ND R R R R R R MS ND ND ND R R S
Note: ‘*’ marked bacteria are Gram-positives and the rest are Gram-negatives. R: Resistant; S: Sensitive; MS: moderately sensitive; ND: not
done. Antibiotics (lg/disc), Ac: amikacin 30; Ak: amoxyclav 30; Am: ampicillin 10; Cd: clindamycin 2; Cf: cefpodoxime 10; Ch: chloram-
phenicol 30; Cot: co-trimoxazole 25; Ce: ceftriaxone 30; Ge: gentamicin 10; Gt: gatifloxacin 5; Nx: norfloxacin 10; Le: levofloxacin 5; Lz:
linezolid 30; Of: ofloxacin 5; Ox: oxacillin 1; Pt: piperacillin/tazobactam 100/10; Tei: teicoplanin 5; Va: vancomycin 30.

against any bacterium (Table 3). The A. acuminata leaf extract 3.4. MIC and MBC of plant extracts
registered the highest value of inhibition zone of 27 mm,
against S. aureus and the lowest value of 20 mm against P. mir- The methanol leaf extract of A. acuminata had the lowest MIC
abilis was recorded; but, inhibition zone values due to A. value, 0.29 mg/ml and the lowest MBC value 0.67 mg/ml
acuminata were recorded for other given bacteria (mm): E. fae- against S. aureus; MIC value of 0.67 mg/ml and MBC value
calis (24), A. baumannii (23), C. freundii (22), E. aerogenes (21), of 1.51 mg/ml against E. faecalis, K. pneumoniae and P. aerug-
E. coli (22), K. oxytoca (23), K. pneumoniae (24), P. vulgaris inosa, while MIC value of 1.51 mg/ml and MBC value of
(21) and P. aeruginosa (25). Thus, A. acuminata extract was 3.41 mg/ml against A. baumannii and K. oxytoca were recorded
effectively capable of controlling all the 11 MDR bacteria. by A. acuminata. On the other hand, the highest MIC value of
Methanol leaf extract of A. indica had shown the highest 3.41 mg/ml, and the highest MBC value of 4.27 mg/ml due to
inhibition-zone size of 22 mm against E. aerogenes, while the A. acuminata extract were noted for C. freundii, E. aerogenes,
lowest value was 12 mm against P. mirabilis. Methanol leaf E. coli, P. mirabilis and P. vulgaris. The methanol leaf extract
extract of P. granatum had the highest value of inhibition- of P. granatum showed the lowest MIC value of 0.29 mg/ml
zone size, 26 mm against S. aureus and the lowest value of and the lowest MBC value of 0.67 mg/ml against S. aureus;
17 mm was against P. mirabilis; the extract was effectively cap- MIC value of 0.67 mg/ml and MBC value of 1.51 mg/ml was
able of controlling all the 11 MDR pathogens by registering noted against C. freundii, E. coli and P. aeruginosa; MIC value
values of inhibition zones ranging from 18 to 25 mm. The of 1.51 mg/ml and MBC value of 3.41 mg/ml was recorded
highest value of inhibition-zone size of 25 mm against S. aur- against E. faecalis; MIC value of 3.41 mg/ml and MBC value
eus and the lowest value of 17 mm against E. aerogenes had of 4.27 mg/ml were recorded against A. baumannii, E. aeroge-
been noted due to the methanol extract of S. febrifuga; and nes, K. oxytoca and K. pneumoniae. The highest MIC value of
20, 23, 21, 19, 22, 21, 19, 20 and 28 mm values of size of zone 4.27 mg/ml and MBC value of 9.63 mg/ml by the extract of
of inhibition were recorded against the pathogenic bacteria, P. granatum were noted against P. mirabilis and P. vulgaris.
E. faecalis, A. baumannii, C. freundii, E. coli, K. oxytoca, K. The methanol leaf extract of S. febrifuga showed the lowest
pneumoniae, P. mirabilis, P. vulgaris and P. aeruginosa, respec- MIC value of 0.67 mg/ml and the lowest MBC value of
tively. The methanol leaf extract of S. febrifuga had an effec- 1.51 mg/ml against S. aureus. MIC value of 1.51 mg/ml and
tive controlling capacity over all the pathogens. Methanol MBC value of 3.41 mg/ml were recorded against A. baumannii.
leaf extracts of the rest 6 plants had moderate control capacity By the extract of S. febrifuga, MIC value 3.41 mg/ml and MBC
on all bacterial strains (Table 3). The total size of zone of inhi- value 4.27 mg/ml were noted against E. faecalis, C. freundii, K.
bition of all used plants is arranged according to the decreasing oxytoca, K. pneumoniae and P. vulgaris and the highest MIC
order, A. acuminata > P. granatum > S. febrifuga > value of 4.27 mg/ml and MBC value of 9.63 mg/ml were
A. indica > B. variegata > T. terrestris > T. cordifolia > recorded against E. aerogenes, E. coli, P. mirabilis and P.
T. chebula > B. diffusa. Moreover, Kruskal–Wallis H test aeruginosa. The MIC and MBC values of leaf extracts of rest
for data of zone of inhibition of the 3 best plants, A. acumi- other plants were recorded (Table 4). A lower MIC/MBC
nata, P. granatum and S. febrifuga yielded the H-value of value signifies that a minimum amount of plant extract is used,
0.83, which was statistically not significant; so, these 3 plants whereas, a higher value signifies the use of comparatively more
were inferred as equally effective for antibacterial activity. amount of plant extract for the control of any bacterium.
Antibacterial activity of medicinal plants against MDR bacteria 89

Table 3 Antibacterial activity as size of zone of inhibition due to 9 selected medicinal plants against bacteria with gentamicin 30 lg/ml
as the positive control.
Bacteria Size of zone of inhibition by plants (Nos. 1 to 9) methanol extracts (mm)
A. acuminata A. indica B. variegata B. diffusa P. granatum S. febrifuga T. chebula T. cordifolia T. terrestris Ge 30 lg/ml
E. faecalis 24 18 17 15 23 20 18 17 19 25
S. aureus 27 20 21 17 26 25 23 19 21 28
A. baumannii 23 17 14 13 22 23 15 – 15 20
C. freundii 22 21 13 12 24 21 – 13 13 21
E. aerogenes 21 22 15 14 20 17 – 14 – 23
E. coli 22 19 19 10 25 19 14 16 17 26
K. oxytoca 23 15 15 13 21 22 15 15 16 22
K. pneumoniae 24 17 17 15 18 21 13 13 15 20
P. mirabilis 20 12 12 – 17 19 11 11 18 22
P. vulgaris 21 13 14 – 19 20 – – 17 23
P. aeruginosa 25 15 20 15 24 18 16 18 20 26
Total zone size 252 189 177 124 239 225 125 136 171
Note: Numbers 1 to 9 are serial numbers of plants given in Table 1; Ge, gentamicin. Values are measurements of zone of inhibition due to
methanol-extracts. ‘‘–’’ sign denotes no activity. Kruskal–Wallis H test for data of zone of inhibition of 3 plants, A. acuminata, P. granatum and
S. febrifuga yielded the H-value of 0.83, which was statistically not significant; so, these 3 plants were equally effective for antimicrobial activity.
The rest other 6 plants were clearly lesser in antimicrobial activity in comparison to cited 3 plants. It was confirmed that 10% DMSO had no
inhibitory effect on any bacterium.

3.5. Qualitative phytochemical analyses had control capacity on all the 11 strains of MDR bacteria.
Moreover, among these 3 best plants in the control of MDR
Qualitative phytochemical analyses were done for methanol bacteria in vitro were A. acuminata, P. granatum and S. febri-
leaf extracts. Phytochemicals, alkaloids, flavonoids, carbohy- fuga. P. granatum has stigmasterol, a sterol with the drug-
drates, terpenoids, steroids, tannins, resins, saponins and likeness score, 0.73 while, S. febrifuga has luteolin-7-O-
anthraquinones, which could be attributed to the recorded sig- glucoside, a flavonoid with the drug-likeness score, 0.86.
nificant antibacterial activities in most extracts (Table 5). From Further work on antibacterial bioactive compounds of
PubChem database, structure and related information of one A. acuminata is in progress, as it had significant microbial
leading antimicrobial compound of each plant was presented activity. However, the plant, T. terrestris has the famous
(Table 6). From previous studies, the antibacterial nature of antimicrobial agent, quercetin, which has an effective drug
these cited 9 compounds were ascertained (Table 6). The likeness score of 0.93; but this plant did not have the best
Molsoft tool (http://molsoft.com/mprop/) was used to find antibacterial activity, in this study or with these bacteria.
out the drug likeness scores of each compound, according to Antibiotic sensitive pathogens have a limited capacity of
their structure canonical ‘simplified molecular-input line-entry virulence as the employed antibiotic controls them in vivo. At
system’ (SMILES). Thus theoretically, based on the available a particular density, the host defense system too helps control
data on drug likeness scores, phytochemicals could be arranged pathogens, when the later are in a limiting number. As known,
in the decreasing order of drug-likeness scores mentioned for the internal protection, antibiotic producing organisms
against: quercetin (0.93) > berberin (0.91) > luteolin-7-O- harbour antibiotic resistant genes in plasmids and chromo-
glugoside (0.86) > kaempferol (0.77) > ursolic acid (0.65) > somes, as well as the transfer mechanisms remain active
argungenin (0.61) and mahmoodin (0.61) > conocarpan (Mamelli et al., 2009). Therefore, such genes and/or trans-
(0.13) and dihydrodedrodehydrodiconiferylalcohol (0.13) > poson are taken up, horizontally by the susceptible group of
anolignan B ( 0.78). It could be inferred here that S. febrifuga bacteria, through bacterial transformation and/or conjugation
among the 3 best plants is the most leading plant with luteolin- (Pages et al., 2008; Warnes et al., 2012).
7-O-glugoside, which has a good score of drug-likeness Moreover, bacteria having simple genomes undergo intrin-
(Table 6). sic (mutations) or acquired genetic (conjugations and transfor-
mation) changes in the presence of an antibiotic, as a stress
factor (Groisman and Ochman, 1996). As a result, accrual
4. Discussion antibiotic resistance mechanisms are the clinical determinants
of the pathogenesis. It had been known that in areas, where a
All of these 9 plants were used by an ethnic tribe, the Kandha particular group of antibiotics are used bacteria resistance to
tribe of Kalahandi district, since time immemorial for primary same antibiotics were in higher numbers (Shrestha et al.,
healthcare needs specifically for infectious diseases. It was seen 2002). Indeed, the horizontal transfer of genetic materials from
that, 11 bacteria isolated from urine samples were resistant to one organism to another appears faster than mutational
the following: aminoglycosides, b-lactams (amoxyclav and changes, a phenomenon popularly called as ‘evolution of quan-
ampicillin), two cephalosporins (ceftriaxone and cefpodoxime) tum leaps’ (Groisman and Ochman, 1996). Progressively, the
as well as, chloramphenicol, signifying most bacterial strains as use of more antibiotics even of higher generations for the con-
resistant to most antibiotics. Additionally, plants, A. acumi- trol of infectious diseases have led to multiple resistances, i.e.,
nata, A. indica, B. variegata, P. granatum and S. febrifuga too many antibiotics are ineffective to progressively increasing
 90
Table 4 MIC and MBC values of selected 9 medicinal plants and of gentamicin as the positive control against bacteria.
Bacterium MIC and MBC values by methanol extracts of 9 plants (mg/ml) Gentamicin lg/ml
1 2 3 4 5 6 7 8 9
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
E. faecalis 0.67 1.51 3.41 4.27 4.27 9.63 9.63 21.67 1.51 3.41 3.41 4.27 4.27 9.63 4.27 9.63 4.27 9.63 0.93 1.87
S. aureus 0.29 0.67 3.41 4.27 3.41 4.27 4.27 9.63 0.29 0.67 0.67 1.51 1.51 3.41 4.27 9.63 3.41 4.27 0.46 0.93
A. baumannii 1.51 3.41 4.27 9.63 9.63 21.67 9.63 21.67 3.41 4.27 1.51 3.41 9.63 21.67 – – 9.63 21.67 3.75 7.50
C. freundii 3.41 4.27 3.41 4.27 9.63 21.67 9.63 21.67 0.67 1.51 3.41 4.27 – – 9.63 21.67 9.63 21.67 1.87 3.75
E. aerogenes 3.41 4.27 3.41 4.27 9.63 21.67 9.63 21.67 3.41 4.27 4.27 9.63 – – 9.63 21.67 – – 0.93 1.87
E. coli 3.41 4.27 4.27 9.63 4.27 9.63 – – 0.67 1.51 4.27 9.63 9.63 21.67 4.27 9.63 4.27 9.63 1.87 3.75
K. oxytoca 1.51 3.41 9.63 21.67 9.63 21.67 9.63 21.67 3.41 4.27 3.41 4.27 9.63 21.67 9.63 21.67 4.27 9.63 0.93 1.87
K. pneumoniae 0.67 1.51 4.27 9.63 4.27 9.63 9.63 21.67 3.41 4.27 3.41 4.27 9.63 21.67 9.63 21.67 9.63 21.67 1.87 3.75
P. mirabilis 3.41 4.27 9.63 21.67 9.63 21.67 – – 4.27 9.63 4.27 9.63 9.63 21.67 9.63 21.67 4.27 9.63 3.75 7.50
P. vulgaris 3.41 4.27 9.63 21.67 9.63 21.67 – – 4.27 9.63 3.41 4.27 – – – – 4.27 9.63 1.87 3.75
P. aeruginosa 0.67 1.51 9.63 21.67 3.41 4.27 9.63 21.67 0.67 1.51 4.27 9.63 9.63 21.67 4.27 9.63 3.41 4.27 0.46 0.93
Note: Numbers 1 to 9 are serial numbers of plants given in Table 1. Values are measurements of MIC and MBC due to methanol extracts. ‘‘–’’ sign denotes no activity; Gentamicin was used as
dilutions from 30 lg/ml.

M.P. Mishra et al.

Antibacterial activity of medicinal plants against MDR bacteria 91

Table 5 Qualitative phytochemical analyses of methanol extracts of 9 medicinal plants.

Sl.No. Plants Alkaloids Resins Glycosides Terpenoids Carbohydrates Saponins Tannins Flavonoids Steroids Anthraquinones
1 A. acuminata + + + + + + + + +
2 A. indica + + + + + +
3 B. variegata + + + + + + + +
4 B. diffusa + + + + +
5 P. granatum + + + + + + + +
6 S. febrifuga + + + + + + + + +
7 T. chebula + + + + + + + +
8 T. cordifolia + + + + +
9 T. terrestris + +
Note: ‘‘+’’ sign denotes presence, and ‘‘ ‘‘sign denotes absence of the compound in a plant.

resistant strains of pathogens, as if growth and momentum geographical zone; rather, along with the same antibiotic the
gained by a descending snow-ball, during the passage of time introduction of complementary or adjuvant drug could be
by mutation and acquisition of genes from related/unrelated aimed, when considered with contemplation the problem of
bacteria, ending in shockingly repellant multidrug bacterial morbidity/mortality from infections due to MDR bacteria
resistance. Older antibiotics slowly become moribund, even (Davies and Davies, 2010).
the resistant mechanism against those are found in certain bac- Applied antibiotics, being of microbial origin, are readily
teria for which, those antibiotics were never applied. Drug won over by pathogenic microbes in vivo. The cell producing
resistant bacteria gain the capability of surviving and multiply- an antibiotic has the characters of the self-protective mecha-
ing under antibiotic-stress conditions, confirming the biological nism as characters/genes, which direct the modes of resistance
rule, ‘any limiting condition for the majority would be an excel- such as, alteration in the cell membrane by efflux mechanism
lent opportunity for the minority’. In the presence of a drug in a or production of external enzymes like, b-Lactamases are
body in vivo, the progeny of a drug sensitive strain is eliminated intrinsically transmitted to similar bacteria, as discussed
and the resistant strain survives, multiplies as if, developing (Rout et al., 2014). In short, suitable antibiotics are required
from a doppelgänger, and predominates ultimately in causing in colossal scale globally, which would give a way to the
a characteristic pathogenesis. It is because a suitable emulating chance of development of resistant strain(s) of pathogenic
agent for the control is absent, and if plant-based antimicrobial bacteria in a Darwinian way, further. Indeed, the present
would be present in parallel along with the employed antibiotic, methodology of methanol-extraction of phytochemicals is a
there would be the coveted blithesome result, since no bac- unique approach, as this solvent helps extraction of most polar
terium how much genetically well-equipped be it may as in a to non-polar phytocompounds (Rezaie et al., 2015). The
cohort of MDR bacteria, can never over-ride complexities of b-Lactam group of antibiotics consisting of penicillins, cepha-
phytochemicals for survival. This fact is repeatedly seen losporins, monobactams, glycopeptides and penems target the
in vitro with several plant extracts (Dubey and Padhy, 2013; peptidoglycan biosynthesis of bacteria. Tetracyclines,
Mishra and Padhy, 2013; Rath and Padhy, 2013; Sahu et al., aminoglycosides, macrolides, lincosamides, streptogramins,
2015). Thus, those in a coalesced manner, as in a crude extract, oxazolidinone (linezolid) and phenicols cause a thwart to the
have a combined controlling effect. translation process in the parasitic cell; quinolones inhibit
It has been demonstrated with Salmonella enterica serotype the DNA replication. Pyrimidines and sulphonamides alter
typhimurium (Alekshun and Levy, 1999). Moreover, MDR carbon metabolism during inhibition of parasitic growth;
Neisseria gonorrheae had been known to acquire ‘MTR and and the most recent ones such as, daptomycin and colistin
SAP A MDR’ systems of genes, from S. enterica serotype inhibit cell membrane functions (see, Davies and Davies,
typhimurium (Hagman and Shaferm, 1995). Discovery and 2010). However, the first effective antimicrobials were the
development of antibiotics in the last century have not only sulphonamides, which have been amply lent themselves for
saved countless human lives, but have provided assurances in further uses in the drug development process as antibacterials
clinical management all over. But, concomitant development in the last several decades. Thus, the concept of complemen-
of antibiotic-resistance mainly in bacteria has dismayed both tary use of phytocompounds along with main stream drugs,
preventive and therapeutic potencies of antibiotics today. In the antibiotics has immersed when the myriad mechanisms of
the odyssey of drug development, antibiotics are introduced drug resistance is considered in the face of success of phyto-
continually and a few of them are modified suiting to the need compounds as non-microbial antimicrobials (Sahu et al., 2015).
to overcome bacterial resistance. Eventually, today there are a Our school has screened out about 250 plants in the last
large number of antibiotics in use. The demand for newer 4 years (Rath and Padhy, 2012; Rath et al., 2012; Dubey and
antibiotics for MDR bacteria in colossal scale has arisen, Padhy, 2012; Mishra and Padhy, 2013; Rath and Padhy,
which has become difficult to meet, as these small molecules 2014; Sahu et al., 2015), using mainly ethanol and water as
are extremely complex in functionality linked to chemical extracting solvents. Among them for 47 plants methanol was
structure. Secondly, an antibiotic ensconced for a typical set used as the solvent (Mishra and Padhy, 2013; Rath and
of infections cannot ordinarily be abandoned as an obsolete Padhy, 2014). A comparative account of MIC values of the
drug, due to reports of dogmatic/realistic resistance in a best plants among 47 against MDR strains of 8 gruesome
92 M.P. Mishra et al.

Table 6 Leading antimicrobial phytochemicals structure, information with properties.

Plant name Leading antimicrobial phytochemical structure Information and properties Drug- References
of leading phytochemical likeness
score

Anolignan B (oc)
Molecular weight: 266.33432 [g/mol]
Molecular formula: C18H18O2
0.78
XLogP3-AA: 5.5
H-Bond donor: 2
H-Bond acceptor: 2

Conocarpan (oc)
Compound ID: 6474521
Rimando
Molecular Weight: 266.33432 [g/mol]
et al.
Molecular Formula: C18H18O2 0.13
A. acuminata (1994a,b),
XLogP3-AA: 4.4
Eldeen et al.
H-Bond donor: 1
(2006)
H-Bond acceptor: 2

Dihydrodehydrodiconiferylalcohol (oc)
Compound ID: 5274623
Molecular weight: 360.40096 [g/mol]
Molecular formula: C20H24O6 0.13
XLogP3-AA: 2.1
H-Bond donor: 3
H-Bond acceptor: 6

Mahmoodin (l)
Compound ID: 126566
Molecular weight: 526.61792 [g/mol]
Siddiqui
A. indica Molecular formula: C30H38O8 0.61
et al. (1992)
XLogP3-AA: 3.7
H-Bond donor: 1
H-Bond acceptor: 8

Kaempferol (f)
Compound ID: 5280863
Molecular weight: 286.2363 [g/mol]
Holler et al.
B. variegata Molecular formula: C15H10O6 0.77
(2012)
XLogP3: 1.9
H-Bond donor: 4
H-Bond acceptor: 6

Ursolic acid (t)

Compound ID: 64945
Molecular weight: 456.70032 [g/mol] Jiménez-
B. diffusa Molecular formula: C30H48O3 0.65 Arellanes
XLogP3-AA: 7.3 et al. (2013)
H-Bond donor: 2
H-Bond acceptor: 3
Antibacterial activity of medicinal plants against MDR bacteria 93

Table 6 (continued)
Plant name Leading antimicrobial phytochemical structure Information and properties Drug- References
of leading phytochemical likeness
score

Stigmasterol (s)
Compound ID: 5280794
Molecular weight: 412.69082 [g/mol]
Awouafack
P. granatum Molecular formula: C29H48O 0.73
et al. (2013)
XLogP3-AA: 8.6
H-Bond donor: 1
H-Bond acceptor: 1

Luteolin-7-O-glucoside (f)
Compound ID: 5291488
Molecular weight: 448.3769 [g/mol]
Khatkar
S. febrifuga Molecular formula: C21H20O11 0.86
et al. (2014)
XLogP3-AA: 0.5
H-Bond donor: 7
H-Bond acceptor: 11

Arjungenin (t)
Compound ID: 12444386
Molecular weight: 504.69852 [g/mol]
Manosroi
T. chebula Molecular formula: C30H48O6 0.61
et al. (2013)
XLogP3-AA: 4.5
H-Bond donor: 5
H-Bond acceptor: 6

Berberin (a)
Compound ID: 2353
Molecular weight: 336.36122 [g/mol]
Choudhary
T. cordifolia Molecular formula: C20H18NO+ 4 0.91
et al. (2013)
XLogP3-AA: 3.6
H-Bond donor: 0
H-Bond acceptor: 4

Quercetin (f)
Compound ID: 5280343
Molecular weight: 302.2357 [g/mol] Rashed and
T. terrestris Molecular formula: C15H10O7 0.93 Butnariu
XLogP3: 1.5 (2014)
H-Bond donor: 5
H-Bond acceptor: 7

Note: a, alkaloid; f, flavonoid; l, limonoid; oc, organic compound; s, sterol; t, terpene.

bacteria is considered, along with the present 3 best plants Cinnamomum tamala, A. acuminata, P. granatum and S. febri-
(Table 7). It is discernible that methanol extracts of most fuga, which had comparatively lower MIC values, as 1.51 or
plants had MIC values as 3.41 or more, except those of 0.67 or 0.29 mg/ml.
94 M.P. Mishra et al.

Table 7 MIC values (mg/ml) of methanol leaf-extracts of plants against pathogenic bacteria.
Bacteria Ef Sa Ab Cf Ea Ec Kp Pa References
Plants
Allium sativum NE 9.63 9.63 4.27 3.41 NE 9.63 NE Rath and Padhy (2014)
Amomum aromaticum 3.41 4.27 NE NE 9.63 NE NE 4.27 Rath and Padhy (2014)
Artocarpus heterophyllus 9.63 9.63 9.63 9.63 9.63 NE 9.63 9.63 Mishra and Padhy (2013)
Cinnamomum tamala 3.41 1.51 NE NE 3.41 NE 9.63 4.27 Rath and Padhy (2014)
Dalbergia latifolia 9.63 3.41 9.63 9.63 9.63 9.63 NE 4.27 Mishra and Padhy (2013)
Gmelina arborea 9.63 3.41 9.63 9.63 NE 9.63 9.63 9.63 Mishra and Padhy (2013)
Melia azedarach 9.63 3.41 9.63 NE NE NE NE 9.63 Mishra and Padhy (2013)
Mentha spicata NE 9.63 9.63 NE 1.51 NE 3.41 4.27 Rath and Padhy (2014)
Mimusops elengi 4.27 4.27 9.63 NE 9.63 9.63 9.63 9.63 Mishra and Padhy (2013)
Myristica fragrans 3.41 NE NE 4.27 3.41 NE 9.63 9.63 Rath and Padhy (2014)
Pongamia pinnata 9.63 4.27 NE NE 9.63 NE NE 9.63 Mishra and Padhy (2013)
Pterocarpus marsupium 4.27 4.27 9.63 9.63 9.63 9.63 NE 9.63 Mishra and Padhy (2013)
Shorea robusta 9.63 4.27 9.63 9.63 NE NE 9.63 3.41 Mishra and Padhy (2013)
Anogeissus acuminata 0.67 0.29 1.51 3.41 3.41 3.41 0.67 0.67 Present work
Punica granatum 1.51 0.29 3.41 0.67 3.41 0.67 3.41 0.67 Present work
Soymida febrifuga 3.41 0.67 1.51 3.41 4.27 4.27 3.41 4.27 Present work
Note: Ef, E. faecalis; Sa, S. aureus; Ab, A. baumannii; Cf, C. freundii; Ea, E. aerogenes; Ec, E. coli; Kp, K. pneumoniae; Pa, P. aeruginosa, ND,
not done; NE, no effect.

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