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●

Research Article

Antibacterial efficacy of five medicinal plants against multidrug-resistant enteropathogenic bacteria infecting under-5 hospitalized children Shakti Rath1,2, Rabindra N. Padhy1,2 1. Department of Botany and Biotechnology, B.J.B. Autonomous College, Bhubaneswar 751014, Odisha, India 2. Central Research Laboratory, IMS & Sum Hospital Medical College, Siksha ‘O’ Anusandhan University, Kalinga Nagar, Bhubaneswar 751003, Odisha, India ABSTRACT OBJECTIVE: To evaluate in vitro antibacterial effectiveness of five medicinal plants used by an Indian aborigine, against 8 multidrug-resistant (MDR) enteropathogenic bacteria isolated from clinical samples of under-5 hospitalized children. METHODS: Antibiotic sensitivity patterns of eight clinically isolated strains of enteropathogenic bacteria, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Salmonella paratyphi, S. typhi, Shigella dysenteriae, S. sonnei and Vibrio cholerae were assessed by disc-diffusion method. Antibacterial activities of 8 solvent-extracts of leaves and bark of five medicinal plants were monitored by the agar-well diffusion method. The microbroth dilution method was used to assess minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Qualitative phytochemical analyses of active plant extracts were carried out. RESULTS: Ethanol, ethyl acetate and methanol extracts of Holarrhena antidysenterica leaf tissue were most effective against 8 MDR pathogens in vitro. Similarly, acetone, ethanol and methanol extracts of Terminalia alata leaf tissue; chloroform, ethyl acetate and methanol extracts of Terminalia arjuna leaf tissue and ethyl acetate, ethanol and methanol extracts of Paederia foetida leaf tissue were most effective in inhibiting in vitro growth of the 8 MDR enteropathogens. Ethyl acetate and methanol extracts of H. antidysenterica bark tissue; acetone, ethanol and methanol extracts of T. alata bark tissue and acetone, ethanol and methanol extracts of T. arjuna bark tissue were most effective in controlling enteropathogen growth. The minimum inhibitory concentration and minimum bactericidal concentration values of the 3 most antimicrobial leaf and bark extracts from the five plants were in the range of 1.56 to 50 mg/mL. CONCLUSION: These 5 plants exhibited in vitro control over a cohort of 8 enteropathogenic bacterial strains isolated from clinical samples. Keywords: ethnomedicinal plants; multidrug resistance; enteropathogenic bacteria; antibacterial assay; phytochemical analysis; plants, medicinal Citation: Rath S, Padhy RN. Antibacterial efficacy of five medicinal plants against multidrug-resistant enteropathogenic bacteria infecting under-5 hospitalized children. J Integr Med. 2015; 13(1): 45–57.


http://dx.doi.org/10.1016/S2095-4964(15)60154-6 Received April 21, 2014; accepted August 15, 2014. Correspondence: Prof. Rabindra N. Padhy; Tel: +91-674-2432034, +91-9437134982; E-mail: [email protected], rnpadhy54@ gmail.com

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1 Introduction

Enteric fever in Cambodian children was dominated by Salmonella enterica, which was resistant to chloramphenicol and trimethoprim-sulfamethoxazole[9]. Bangladesh, with large areas of lowlands/swamps, was the epicentre of an enteric fever outbreak caused by serotypes of S. typhi and S. paratyphi of S. enterica, which were resistant to chloramphenicol, trimethoprim-sulfamethoxazole, nalidixic acid and ampicillin [10]. In a 2009 study of Romanian children suffering from diarrhoea, 61 of the 250 patients were infected with DEC[11]. Shigellosis and salmonellosis were the most prevalent enteric diseases in children of Mozambique, and several serotypes were described with resistance to the most commonly used antibiotics, including trimethoprim-sulfamethoxazole in 84% cases[12]. Following WHO recommendations for the inclusion of herbal compounds as complementary or alternative medicine (CAM), we selected five well-known ethnomedicinal plants, Cassia fistula, Holarrhena antidysenterica, Terminalia alata, Terminalia arjuna and Paederia foetida (Figure 1) to evaluate in vitro antibacterial effects on a cohort of MDR enteropathogenic bacteria isolated from clinical samples of under-5 children suffering from diarrhoea. Several studies have reported the use of herbal preparations for the control of infectious bacteria[13–15]. C. fistula, the famous Indian laburnum, is known for its medicinal use in treatment of abdominal tumours, glands, liver, throat burns, constipation, convulsions, diarrhoea, epilepsy, leprosy, general skin diseases and syphilis. It is found throughout tropical Asia, South Africa, East Africa, Mexico and Brazil[16]. The plant H. antidysenterica has been used for a wide variety of ailments in traditional medicine of India, including for colic and fever, as a carminative, astringent, lithontriptic tonic, aphrodisiac, cardio-suppressant, diuretic and antihypertensive[17]. In Indian Ayurveda, Kutajarishta, a combination of 5 phyto-extracts, including leaves of H. antidysenterica, is prescribed to control diarrhoea and dysentery[18]. Its seeds are used as an anti-diabetic drug in several Asian countries. The in vitro antibacterial activity of H. antidysenterica has been shown, as well as its in vivo ability to control diarrhoea, dysentery, hemorrhage, hemorrhoids, amoebiasis and hepatitis[17]. T. alata is a deciduous tree with grey or black, deeply cracked, and rough bark, native to India, and also found in Indo-China, Myanmar and Thailand. The tree normally grows to a height of 30–35 m. Traditionally, the bark of T. alata has been widely used in Indian ethnomedicine and Ayurveda for a variety of healing purposes[19]. A decoction of the bark is used as cardiotonic, diuretic and styptic, and also to treat boils, bronchitis, diarrhoea, fever, fractures, haemorrhage, pruritus and ulcers; its gum in particular is used as a laxative. To treat diarrhoea or dysentery, about

Enteropathogenic bacteria are the principal causes of high infant and child (under-5) mortality as well as morbidity in all age groups, especially in aged and immunocompromised patients, in developing countries[1]. This group of bacteria includes Vibrio cholerae; its infection can quickly lead to a life-threatening situation due to loss of fluid. Such situations are often addressed by infusion of electrolytes to the blood and the simultaneous antibiotic therapy[2]. However, when the causative pathogen is multidrug-resistant (MDR), the antibiotic therapy is ineffective. Further, enteric infections are often characterized by more than one pathogen such as Escherichia coli and Klebsiella pneumoniae together. Thus, clinical management of these infections can be complicated and unsuccessful[3]. World Health Organization (WHO) records that more than 10 million under-5 children die annually from pneumonia and diarrhoea, with malnutrition being the underlying cause of disease — Asian and African countries contribute more than half of these cases[4]. The mortality rates of under-5 children in Nigeria, the Indian sub-continent and China are the highest in comparison to all other countries. Among Iranian adults and young adults, fluoroquinolone-resistant diarrhoeagenic E. coli (DEC), including enteroaggregative, enteropathogenic, enterotoxigenic and shiga-toxin producing strains, accounted for 21.4% of infections [5]. A recent study from India comprising seven hospitals from New Delhi, Vellore, Bangalore and Ludhiana found that the resistance of enteropathogens to antibiotics was greatest for quinolones, macrolides, aminoglycosides, trimethoprim, β-lactams and sulphonamides of higher generations (in decreasing order)[6]. Further it has been summarized that Gram-negative (GN) enteropathogens (K. pneumoniae, Salmonella typhi, E. coli, S. paratyphi A and O1 strain of V. cholerae) have the highest levels of drug resistance, individually, to tobramycin, amikacin, and cefotaxime; these bacteria were also found to be extended-spectrum β-lactamase (ESBL) positive in 64%, 54%, 32%, 14% and 25% of cases, respectively, in seven hospitals[6]. A Kenyan report found that diarrhoea was the main cause of morbidity and mortality in children in sub-Saharan Africa[7]. Among Kenyan children treated for diarrhoea, 17.7% (of 651 patients) suffered primarily from infection of Shigella, V. cholerae, E. coli and Salmonella. Further, E. coli, and Shigella species were the bacteria which had 80%–100% resistance to the commonly used antibiotics [7]. A study from Nigeria, on the prevalence of Enterobacteriaceae in diarrhoea patients, described E. coli O157 as the causative strain, and found it to be resistant to seven common antibiotics; these drug-resistant diarrhoeagenic bacteria were present in both clinical samples and in water samples[8]. January 2015, Vol.13, No.1

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3–4 teaspoons of fresh bark juice (about 50 g bark crushed in 10 teaspoons of water and filtered) are taken 3 times a day[20]. T. arjuna is a large deciduous tree, growing to a height of 20 to 30 m, found throughout the South Asian region, mainly in India, Burma, Sri Lanka and Mauritius[21]. It is one of the most versatile medicinal plants in traditional Indian medicine, having a wide spectrum of biological activity for therapeutic uses. The bark of T. arjuna is used as an antidysenteric, antipyretic, astringent, anticoagulant, antimicrobial, antiuremic, cardiotonic and lithontriptic agent. Several useful phyto-constituents have been isolated from T. arjuna, which include triterpenoids with cardiovascular-protective properties, as well as tannins and flavonoids having anticancer and antimicrobial properties. Bark-powders are diuretic, help in cirrhosis of liver and give relief in the symptomatic hypertension[21]. P. foetida is a perennial climbing shrub with a strong foul-odour and are well distributed in tropical humid areas of Asia, Africa and South America[22]. Several therapeutic properties have been attributed to P. foetida in Indian and traditional Chinese medicine systems. It is used mainly to treat diarrhoea, dysentery, enteritis, gastritis and stomach ache[22]. In this paper, 8 solvents are used to prepare extracts of leaf and bark tissues from 5 medicinal plants; the ability of these extracts to control growth of 8 enteropathogenic bacteria, isolated from clinical samples of under-5 hospitalised children was evaluated.

2 Materials and methods 2.1 Collection of plant materials Medicinal plants, C. fistula, H. antidysenterica, T. alata, T. arjuna and P. foetida (Figures 1A–1E), were collected from the forests of India’s Eastern mountains, located in the district of Kalahandi, Odisha. About 50 respondents and practitioners of traditional medicine among the Kandha, an aboriginal tribe, were interviewed and the ethnomedicinal information that they shared was documented by the ‘snowball technique’ (Table 1). The plants were identified using local botanical keys and voucher specimens were deposited at the herbarium of the Department of Botany, Government Autonomous College, Bhawanipatna (Table 1)[23]. 2.2 Preparation of plant extracts Mature leaves and bark of plants were separately crushed into powders. Eight solvents in a range from polar to non-polar were selected for use in the study: petroleum ether, ethyl acetate, chloroform, n-hexane, acetone, ethanol, methanol and water. Extracts of powdered bark or leaves were prepared in each solvent by incubating 5 g of powdered plant tissue with 25 mL of the solvent at 4 ℃ for 72 h. Each extract was filtered, and the filtrate was concentrated in a rotary evaporator at 40 ℃ until a sticky mass was obtained. This mass was weighed and dissolved in 1 mL of 10% (v/v) dimethyl sulfoxide (DMSO). Individual solvent extracts of leaves and bark were used to evaluate antibacterial activity in vitro. For each plant sample these

Figure 1 Images of Cassia fistula (A), Holarrhena antidysenterica (B), Terminalia alata (C), Terminalia arjuna (D), and Paederia foetida (E) Journal of Integrative Medicine

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www.jcimjournal.com/jim Table 1 Ethnomedicinal uses of the 5 medicinal plants used Plant name; voucher specimen number

Family name

Local name; parts used

Caesalpiniaceae

Sunari; leaf/bark

Skin diseases, burning sensations, syphilis, boils, leprosy, ringworm infection, dry cough, bronchitis, dysentery and inflammations

Holarrhena antidysenterica L Wall.; Apocynaceae GACB 205

Kutaja; leaf/bark

Diarrhoea and skin diseases, urinary tract infections, amoebic dysentery and respiratory infections

Terminalia alata Heyne ex Roth*; GACB 189

Combretaceae

Sahaj; leaf/bark

Diarrhoea and dysentery

Terminalia arjuna (Roxb.) Wight & Arn; GACB 097

Combretaceae

Arjuna; leaf/bark

Skin diseases, urinary infection, skin aliments including acne and acute diarrhoea

Paederia foetida L.**; GACB 395

Rubiaceae

Prasarini; leaf

Diarrhoea, dysentery, skin sores and tooth infections

Cassia fistula L.; GACB 467

*

Ethnomedicinal uses

**

Synonyms of T. tomentosa, and T. elliptica; Synonyms of P. scandens, P. magnifica, P. tomentosa, and Gentiana scandens. GACB: Government Autonomous College, Bhawanipatna.

steps were repeated and all extracts were stored at 4 ℃ for further use, as detailed[3,24]. 2.3 Isolation and identification of enteropathogenic bacteria Eight enteropathogens (Enterobacter aerogenes, Escherichia coli, K. pneumoniae, S. paratyphi, S. typhi, Shigella dysenteriae, S. sonnei and V. cholerae) were isolated from stool samples of 35 under-5 children from the paediatric ward of IMS and Sum Hospital, Bhubaneswar, over a period of 6 months. All of these patients had been admitted for diarrhoea and related gastrointestinal complaints. Identification and culture of isolated bacterial strains were conducted on specialized media following established protocols [19]. Standard drug-sensitive strains of these bacteria were also obtained from Microbial Type Culture Collection (MTCC), Chandigarh, India and served as reference control strains. 2.4 Determination of antibiotic sensitivity All bacterial strains isolated from patients, as well as reference control strains were subjected to antibiotic sensitivity tests by the disc diffusion/Kirby-Bauer’s method (Figure 2). These experiments used 16 antibiotics from 6 different groups, as recommended by the Clinical and Laboratory Standards Institute Guidelines 2014[25]. The double-disc synergy test was used to determine whether strains produced extended-spectrum beta-lactamase (ESBL), following established protocols[19,26]. 2.5 Evaluation of antibacterial activity The antibacterial activity of each of the 8 solventextracts of leaves and bark was assayed using the agarwell diffusion method [19]. DMSO (10% by), with out antibacterial activity, and chloramphenicol (30 µg/mL), with an average inhibition zone of 20 mm, were used as negative and positive reference controls, respectively. The values of minimum inhibitory concentration (MIC) and January 2015, Vol.13, No.1

Figure 2 Antibiotic sensitivity test by Kirby-Bauer’s method of E. aerogenes on Muller-Hinton agar

minimum bactericidal concentration (MBC) for each plant extract were determined using standard protocols[19]. 2.6 Phytochemical analysis Phytochemical analyses of each extract (both leaves and bark) were conducted to confirm the presence of phytochemicals, reducing sugars, saponins, flavonoids, steroids, terpenoids, tannins, alkaloids, resins and glycosides using standard methods[19]. Experiments were repeated 3 times and the results of the third repeated experiment are presented here. 3 Results 3.1 Ethnomedicinal information Ethnomedicinal use of these 5 medicinal plants was obtained through interviews with aborigines of Kalahandi

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district, Odisha. These data indicated that these plants are mainly used for boils, bronchitis, burning sensations, diarrhoea, dysentery, dry cough, inflammations, leprosy, ringworm infection, syphilis, skin sores and infections including urinary tract infections (Table 1). 3.2 Antibiotic susceptibility of the GN bacterial strains Antibiotic susceptibility tests of the 8 isolated GN bacterial strains were carried out using 16 antibiotics from 6 groups (aminoglycosides, β-lactams, cephalosporins, fluoroquinolones, sulphonamides and 3 synthetic antibiotics). The isolated S. paratyphi strain was resistant to 6 antibiotics: ampicillin, amoxyclav, ceftriaxone, cefpodoxime, gatifloxacin and co-trimoxazole. The S. paratyphi strain was moderately sensitive to ciprofloxacin and fully sensitive to the remaining 8 antibiotics. Likewise, antibiograms of the other 7 strains were recorded (Table 2). All isolated GN bacteria were ESBL producers, as all the isolates were resistant to both cephalosporin antibiotics (Table 2). This proves the organisms used in the study were amply MDR. 3.3 Antibacterial activity of plant extracts The antibacterial activities of each of the 8 solvent extracts were evaluated using the agar-well diffusion method, on separate lawn-cultures of each of the 8 bacterial isolates. Chloroform leaf extract of C. fistula had the largest zone of inhibition, 25 mm, against S. dysenteriae and K. pneumoniae, and 24 mm against S. sonnei. Similarly, its ethanol leaf extract had its largest zone of inhibition, 25 mm, against E. coli and S. paratyphi, next largest, 24 mm, against E. aerogenes and S. typhi, and 23 mm against K. pneumoniae. The petroleum-ether and n-hexane leaf extracts of C. fistula registered very low antibacterial activity compared with the other 6 solvent extracts (Table 3).

Bark extracts had different antibacterial activity compared with leaf extracts using the same solvents. Ethyl acetatebark-extract had its maximum size of zone of inhibition of 23 mm against E. coli; a zone of 21 mm against E. aerogenes, K. pneumoniae, S. typhi and S. sonnei, and a zone of 20 mm against S. paratyphi and S. dysenteriae. Similarly, acetone bark extract of C. fistula registered its maximum size of zone of inhibition of 24 mm against S. typhi. It also had a zone of 22 mm against E. aerogenes and S. paratyphi and a zone of 21 mm against K. pneumoniae, S. dysenteriae and S. sonnei (Table 3). For C. fistula, leaf extracts with chloroform, acetone and ethanol had the greatest antibacterial activity. Bark of the same plant had the greatest antibacterial activity when extracted with chloroform, acetone and ethyl acetate. The zones of inhibition sizes for the remaining 8 solvent extracts of leaves and bark from other 4 medicinal plants were also evaluated using the agar-well diffusion method against 8 MDR enteropathogenic bacteria. Results demonstrated that the ethanol, ethyl acetate and methanol leaf extracts of H. antidysenterica were the most effective against MDR pathogenic strains (Table 4). Similarly, acetone, ethanol and methanol leaf extracts of T. alata (Table 5), chloroform, ethyl acetate and methanol leaf extracts of T. arjuna (Table 6), and ethyl acetate, ethanol and methanol extracts of P. foetida (Table 7) were also highly effective at inhibiting bacterial growth in vitro. Bark of H. antidysenterica extracted in acetone, ethyl acetate and methanol (Table 5, and Figure 3), T. alata extracted in acetone, ethanol and methanol (Table 5), as well as T. arjuna extracted in acetone, ethanol and methanol (Table 6) were all highly effective at inhibiting growth of 8 MDR enteropathogens in vitro.

Table 2 Antibiotic susceptibility pattern of the isolated enteropathogenic bacteria Susceptibility to prescribed antibiotics Bacteria

Aminoglycosides

β-Lactams

Ac

Ge

Am Ak

E. aerogenes

R

R

R

E. coli

R

R

K. pneumoniae

R

S

Cephalosporins

Fluoroquinolones

Sulfonamide

Stand-alones

Pt

Ce

Cf

Ci

Gf

Na

No

Of

Co-t

Ch

Nf

Te

R

R

R

R

S

R

R

R

R

R

S

R

I

R

R

R

R

R

S

R

R

R

R

S

S

R

R

R

R

R

R

R

R

R

R

R

R

R

S

R

R

S. paratyphi

S

S

R

R

S

R

R

S

R

S

S

S

R

S

I

S

S. typhi

R

R

R

R

R

R

R

I

R

R

R

R

R

S

R

R

S. dysenteriae

R

I

R

R

R

R

R

S

R

R

R

R

R

S

R

R

S. sonnei

R

R

R

R

R

R

R

S

R

R

R

R

R

S

R

I

V. cholerae

R

R

S

S

S

R

S

S

S

S

S

S

R

S

S

S

R: resistant; S: sensitive; I: moderately sensitive. Antibiotics (µg/disc): Ac, amikacin 30; Ak, amoxyclav 30; Am, ampicillin 10; Ce, ceftriaxone 30; Cf, cefpodoxime 10; Ch, chloramphenicol 30; Ci, ciprofloxacin 5; Co-t, co-trimoxazole 25; Ge,gentamicin10; Gf, gatifloxacin 5; Na, nalidixic acid 30; Nf, nitrofurantoin 300; No, norfloxacin 10; Of, ofloxacin 5; Pt, piperacillin-tazobactam100-10; Te, tetracycline 30. Journal of Integrative Medicine

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www.jcimjournal.com/jim Table 3 Antibacterial assays by agar-well diffusion method of 8 cold solvent leaf and bark extracts of C. fistula against 8 MDR enteropathogenic bacterial strains (zone of inhibition in mm) MDR bacteria

Petroleum ether

Ethyl acetate

Chloroform

n-Hexane

Acetone

Ethanol

Methanol

Water

Chl

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

E. aerogenes

—

—

17

21

23

21

—

—

24

22

24

18

14

12

12

08

21

E. coli

—

—

19

23

21

20

6

4

21

20

25

19

15

13

13

11

20

K. pneumoniae

—

—

16

21

25

20

—

—

23

21

23

20

14

13

16

4

21

S. paratyphi

—

—

17

20

22

21

7

6

20

22

25

0

19

16

10

2

20

S. typhi

6

8

18

21

21

20

—

—

22

24

24

19

17

13

16

11

22

S. dysenteriae

—

—

14

20

25

21

—

—

23

21

20

16

17

12

13

0

21

S. sonnei

4

7

15

21

24

22

7

5

23

21

21

15

15

12

12

1

20

V. cholerae

—

—

13

15

16

12

—

—

16

13

16

12

12

10

10

06

21

MDR: multidrug resistant; LE: leaf extract; BE: bark extract. Chl: chloramphenicol 30 µg/mL. Table 4 Antibacterial assays by agar-well diffusion method of 8 cold solvent leaf and bark extracts of H. antidysenterica against 8 MDR enteropathogenic bacterial strains (zone of inhibition in mm) MDR bacteria

Petroleum ether

Ethyl acetate

Chloroform

n-Hexane

Acetone

Ethanol

Methanol

Water

Chl

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

E. aerogenes

5

3

25

24

18

19

—

—

17

21

23

15

24

21

13

11

21

E. coli

6

5

21

20

17

15

—

4

19

21

22

16

25

23

12

10

20

K. pneumoniae

—

—

21

22

16

18

—

—

19

20

21

17

23

24

12

10

21

S. paratyphi

—

—

22

20

12

15

—

—

17

23

22

15

20

22

14

13

20

S. typhi

12

9

21

20

15

13

—

3

14

21

24

11

22

20

15

12

22

S. dysenteriae

—

—

24

22

14

12

—

—

12

21

20

12

21

21

12

10

21

S. sonnei

—

—

23

23

16

17

—

—

15

20

22

13

23

20

15

13

20

V. cholerae

—

—

15

13

10

12

—

—

10

13

17

11

14

12

10

7

21

MDR: multidrug resistant; LE: leaf extract; BE: bark extract; Chl: chloramphenicol 30 µg/mL. Table 5 Antibacterial assays by agar-well diffusion method of 8 cold solvent leaf and bark extracts of T. alata against 8 MDR enteropathogenic bacterial strains (zone of inhibition in mm) MDR bacteria

Petroleum ether

Ethyl acetate

Chloroform

n-Hexane

Acetone

Ethanol

Methanol

Water

Chl

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

E. aerogenes

—

—

15

12

14

11

—

—

26

21

24

21

26

22

11

09

21

E. coli

—

—

17

15

15

14

—

—

23

23

22

20

25

23

10

07

20

K. pneumoniae

—

—

19

14

19

16

6

5

21

20

24

20

23

24

13

10

21

S. paratyphi

—

—

16

13

16

12

7

5

20

20

22

23

21

19

14

11

20

S. typhi

—

—

18

14

18

15

—

—

25

21

24

22

23

21

13

10

22

S. dysenteriae

—

—

18

11

12

11

—

—

24

22

20

21

21

21

12

08

21

S. sonnei

—

—

17

15

17

11

8

4

23

20

22

21

23

21

11

12

20

V. cholerae

—

—

10

09

13

—

—

—

16

12

17

15

16

13

07

03

21

MDR: multidrug resistant; LE: leaf extract; BE: bark extract; Chl: chloramphenicol 30 µg/mL. January 2015, Vol.13, No.1

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www.jcimjournal.com/jim Table 6 Antibacterial assay by agar-well diffusion method of 8 cold solvent leaf and bark extracts of T. arjuna against 8 MDR enteropathogenic bacterial strains (zone of inhibition in mm) MDR bacteria

Petroleum ether

Ethyl acetate

Chloroform

n-Hexane

Acetone

Ethanol

Methanol

Water

Chl

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

LE

BE

E. aerogenes

7

4

23

16

21

17

—

—

15

20

17

21

26

23

10

06

21

E. coli

—

—

24

17

24

17

—

—

18

23

15

20

24

21

11

08

20

K. pneumoniae

5

7

21

15

25

15

—

—

14

20

14

20

23

19

14

11

21

S. paratyphi

—

—

20

17

21

16

—

—

19

23

17

22

25

23

17

12

20

S. typhi

—

—

20

16

20

16

—

17

24

12

20

24

21

13

11

22

S. dysenteriae

—

—

24

20

23

14

—

—

15

23

18

22

22

21

11

08

21

S. sonnei

8

5

23

14

21

18

—

—

18

22

14

21

24

20

16

12

20

V. cholerae

—

—

17

12

15

11

—

—

12

16

10

16

16

12

8

5

21

MDR: multidrug resistant; LE: leaf extract; BE: bark extract; Chl: chloramphenicol 30 µg/mL.

Table 7 Antibacterial assays by agar-well diffusion method of 8 cold solvent leaf extracts of P. foetida against 8 MDR enteropathogenic bacterial strains (zone of inhibition in mm) Petroleum ether

Ethyl acetate

Chloroform

n-Hexane

Acetone

Ethanol

Methanol

Water

Chl

E. aerogenes

10

24

17

—

12

25

21

13

21

E. coli

7

20

15

—

11

26

23

10

20

K. pneumoniae

7

22

18

8

10

27

24

12

21

S. paratyphi

8

25

15

7

13

25

22

13

20

S. typhi

12

22

13

5

11

21

20

12

22

S. dysenteriae

9

26

12

4

11

22

21

12

21

S. sonnei

7

23

17

7

10

23

20

13

20

V. cholerae

—

13

12

—

9

17

14

07

21

MDR bacteria

MDR: multidrug resistant; Chl: chloramphenicol 30 µg/mL.

3.4 MIC and MBC values of the active plant extracts Ethanol, chloroform and acetone extracts of C. fistula were carried through to this experiment because they had the highest antibacterial activities. These active solvent extracts were further used to determine the MIC and MBC values against the 8 MDR enteropathogens. The acetone leaf extract had an MIC of 3.125 mg/mL against E. aerogenes, K. pneumoniae, S. typhi, S. dysenteriae and S. sonnei. The same extract had an MIC of 6.25 mg/mL against E. coli, and S. paratyphi and 12.5 mg/mL against V. cholerae (Table 8). Bark of C. fistula extracted in acetone had an MIC of 3.125 mg/mL against E. aerogenes, K. pneumoniae, S. typhi, S. dysenteriae and S. sonnei; 6.25 mg/mL against E. coli, and S. paratyphi and 25 mg/mL against V. cholerae (Table 8). The acetone leaf-extract of C. fistula had an MBC of 12.5 mg/mL against E. aerogenes, K. pneumoniae, S. typhi,

Figure 3 Agar-well diffusion of methanol bark extract of H. antidysenterica against E. aerogenes Journal of Integrative Medicine

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S. dysenteriae and S. sonne, 25.0 mg/mL against E. coli and 50 mg/mL against V. cholerae. Similarly, for acetonebark extracts of C. fistula, an MBC of 12.5 mg/mL against E. aerogenes, K. pneumoniae, S. typhi, S. dysenteriae and S. sonnei; 25 mg/mL against E. coli, and S. paratyphi and 50 mg/mL against V. cholerae (Table 8). The MIC and MBC values for the chloroform and ethanol leaf extract and chloroform and ethyl acetate bark extracts of C. fistula were also determined, and are shown in Table 8. The MIC and MBC values of the three best active leaf and bark extracts for each of the other four plants are shown in (Tables 9–12). 3.5 Phytochemical analysis Qualitative phytochemical analysis of the active leaf and bark extracts of the 5 medicinal plants was carried out. From the analysis of acetone leaf extracts of C. fistula, the presence of alkaloids, glycosides, terpenoids, reducing sugars,

saponins, tannins, flavonoids and absence of steroids were confirmed. Further, from the analysis of acetone bark extract of C. fistula, the presence of alkaloids, glycosides, terpenoids, saponins, tannins, flavonoids and absence of reducing sugars and steroids were confirmed. The same analysis was conducted for the remaining solvent extracts of C. fistula bark and leaf tissue, as well as the other 4 medicinal plants (Table 13). Comparing the zone of inhibition formed around each of the eight MDR enteropathogenic bacteria, petroleum ether and n-hexane plant extracts were the most ineffective solvents of the eight solvent used. Aqueous leaf and bark extracts of all used plants also had relatively low antibacterial activity. Extracts prepared using ethyl acetate, acetone, chloroform, methanol and ethanol exhibited moderate to high antibacterial activities against all MDR strains of enteropathogens. The preliminary phytochemical

Table 8 MIC and MBC values of the best 3 cold bioactive leaf and bark extracts of C. fistula against isolated strains enteropathogenic bacteria (mg/mL) C. fistula leaf extract MDR bacteria

Acetone MIC

MBC

C. fistula bark extract

Chloroform MIC

MBC

Ethanol MIC

MBC 12.5

E. aerogenes

3.125

12.5

3.125

12.5

3.125

E. coli

6.25

25.0

3.125

12.5

1.56

K. pneumoniae

3.125

12.5

1.56

S. paratyphi

6.25

12.5

3.125

12.5 25.0

S. typhi

3.125

12.5

6.25

S. dysenteriae

3.125

12.5

1.56

S. sonnei V. cholerae

3.125 12.50

12.5 50.0

1.56 12.5

6.25

6.25 6.25 25.0

6.25

Acetone MIC

MBC

Chloroform MIC

MBC

Ethyl acetate MIC

MBC

3.125

12.5

6.25

12.5

3.125 12.5

6.25

25.0

6.25

25.0

1.56

3.125

3.125

12.5

3.125

12.5

6.25

12.5

3.125 12.5

1.56

12.5

6.25

25.0

6.25

12.5

6.25

1.56

6.25

6.25

25.0

3.125

12.5

12.5

50.0

25.0

3.125

12.5

6.25

25.0

3.25

12.5

3.125

12.5

6.25

12.5

6.25

25.0

3.125 25.00

12.5 50.0

1.56 25.0

6.25 50.0

3.125 25.0 25.0

50.0

MDR: multidrug resistant; MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration. Table 9 MIC and MBC values of the best 3 cold bioactive leaf and bark extracts of H. antidysenterica against isolated strains enteropathogenic bacteria (mg/mL) H. antidysenterica leaf extract MDR bacteria E. aerogenes

Ethanol

Ethyl acetate

H. antidysenterica bark extract

Methanol

MIC

MBC

MIC

MBC

MIC

MBC

1.56

6.25

1.56

6.25

1.56

6.25

E. coli

3.125

12.5

3.125

12.5

1.56

K. pneumoniae

3.125

25.0

3.125

12.5

3.125

S. paratyphi

3.125

12.5

S. typhi

1.56

6.25

MBC

MIC

MBC

3.125

12.5

1.56

6.25

MIC

MBC

3.125

25.0

3.125

12.5

3.125

25.0

3.125

12.5

6.25

25.0

3.125

25.0

3.125

12.5

12.5

6.25

25.0

1.56

12.5

3.125

12.5

3.125

12.5

12.5

3.125

12.5

3.125

12.5

3.125

25.0

6.25

25.0

6.25

25.0

1.56

3.125

12.5

3.125

25.0

MIC

Methanol

3.125

S. dysenteriae

12.50

Ethyl acetate

3.125

S. sonnei V. cholerae

6.25 12.5

Acetone

12.5

6.25 12.5 50.0

3.125

12.5

3.125

12.5

3.125

12.5

6.25

25.0

3.125

12.5

6.25

25.0

3.125

12.5

6.25

25.0

12.50

50.0

25.0

25.0

25.0

50.0

25.0

50.0

MDR: multidrug resistant; MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration. January 2015, Vol.13, No.1

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www.jcimjournal.com/jim Table 10 MIC and MBC values of the best 3 cold bioactive leaf and bark extracts of T. alata against isolated strains of enteropathogenic bacteria (mg/mL) T. alata leaf extract MDR bacteria E. aerogenes

Acetone

Ethanol

MIC

MBC

MIC

MBC

MIC

MBC

1.56

6.25

1.56

6.25

1.56

6.25

E. coli

3.125

12.5

3.125

K. pneumoniae

3.125

12.5

1.56

S. paratyphi

6.25

25.0

S. typhi

1.56

S. dysenteriae

1.56

S. sonnei

3.125

V. cholerae

T. alata bark extract Methanol

12.5

12.5 6.25

3.125

6.25

25.0

1.56

6.25 25.0 25.0

6.25

1.56

6.25

3.125

12.5

Acetone

Ethanol

Methanol

MIC

MBC

MIC

MBC

MIC

MBC

3.125

12.5

3.125

12.5

3.125

12.5 12.5

3.125

12.5

6.25

25.0

3.125

3.125

12.5

6.25

12.5

1.56

6.25

3.125

12.5

6.25

25.0

3.125

12.5

6.25

25.0

3.125

12.5

6.25

25.0

3.125

25.0

3.125

12.5

6.25

25.0

3.125

12.5

3.125

12.5

3.125

12.5

3.125

12.5

3.125

12.5

3.125

12.5

6.25

25.0

3.125

25.0

3.125

12.5

12.5

50.0

12.5

50.0

25.0

50.0

25.0

50.0

25.0

50.0

MDR: multidrug resistant; MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration. Table 11 MIC and MBC values of the best 3 cold bioactive leaf and bark extracts of T. arjuna against isolated strains of enteropathogenic bacteria (mg/mL) T. arjuna leaf extract MDR bacteria

Chloroform

Ethyl acetate

T. arjuna bark extract Methanol

Acetone

Ethanol

Methanol

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

E. aerogenes

3.125

12.5

3.125

12.5

1.56

6.25

6.25

25.0

3.125

12.5

3.125

E. coli

1.56

6.25

1.56

6.25

3.125

12.5

6.25

25.0

3.125

K. pneumoniae

1.56

6.25

3.125

12.5

3.125

6.25

25.0

6.25

25.0

6.25

25.0

S. paratyphi

3.125

12.5

6.25

25.0

1.56

3.125

12.5

3.125

12.5

3.125

12.5

S. typhi

6.25

25.0

3.125

12.5

3.125

12.5

1.56

12.5

6.25

25.0

3.125

12.5

S. dysenteriae

3.125

12.5

1.56

12.5

3.125

25.0

3.125

25.0

3.125

25.0

3.125

25.0

S. sonnei

3.125

12.5

3.125

25.0

1.56

3.125

12.5

3.125

12.5

6.25

25.5

50

12.5

V. cholerae

25.0

50

6.25

12.5

1.56

12.5 6.25

6.25 25.0

12.5

50

25.0

50

MBC

25.0

6.25 6.25

50.0

MDR: multidrug resistant; MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration. Table 12 MIC and MBC values of the best 3 cold bioactive leaf extracts of P. foetida against isolated strains enteropathogenic bacteria (mg/mL) P. foetida leaf extract Ethyl acetate

MDR bacteria E. aerogenes

Methanol

MIC

MBC

MIC

MBC

MIC

MBC

1.56

6.25

1.56

6.25

6.25

12.5

E. coli

6.25

K. pneumoniae

3.125

S. paratyphi

1.56

S. typhi

3.125

S. dysenteriae

1.56

S. sonnei

3.125

V. cholerae

Ethanol

12.5

12.5 6.25 6.25 12.5 6.25 12.5 25.0

1.56

12.5

3.125

1.56

6.25

3.125

1.56 3.125

6.25 12.5

1.56

6.25 12.5 6.25

6.25

25.0

3.125

25.0

6.25

12.5

3.125

12.5

6.25

12.5

12.5

50.0

12.5

25.0

MDR: multidrug resistant; MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration. Journal of Integrative Medicine

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www.jcimjournal.com/jim Table 13 Phytochemical analysis of the active extracts of 5 medicinal plants Plant part

Solvent extracts Alkaloids Glycosides Terpenoids Reducing sugars Saponins Tannins Flavonoids Steroids

Cassia fistula Leaves

Bark

Acetone

+

+

+

+

+

+

+

–

Chloroform

+

–

+

–

+

+

+

–

Ethanol

+

+

+

+

+

+

+

+

Acetone

+

+

+

–

+

+

+

–

Chloroform

+

–

+

+

+

+

+

+

EA

+

+

+

+

–

+

+

–

Ethanol

+

–

+

+

–

+

+

–

EA

+

+

+

+

+

+

+

+

Methanol

+

+

+

+

+

+

+

+

Holarrhena antidysenterica Leaves

Bark

Acetone

+

+

+

+

+

+

+

–

EA

+

–

+

–

+

+

+

–

Methanol

+

–

+

+

+

+

+

–

+

+

+

+

+

+

+

–

Terminalia alata Leaves

Bark

Acetone Ethanol

+

+

+

+

+

+

+

–

Methanol

+

+

+

+

+

+

+

–

Acetone

+

+

+

+

–

+

+

–

Ethanol

+

–

+

–

+

+

+

–

Methanol

+

+

+

+

–

+

+

+

Terminalia arjuna Leaves

Bark

Chloroform

+

–

+

+

+

+

+

+

EA

+

+

+

+

–

+

+

+

Methanol

+

+

+

+

–

+

+

–

Acetone

+

+

+

+

+

+

+

–

Ethanol

+

+

+

+

+

+

+

–

Methanol

+

–

+

+

+

+

+

–

Paederia foetida Leaves

EA

+

+

+

–

–

+

+

–

Ethanol

+

+

+

+

+

+

+

+

Methanol

+

–

+

+

+

+

+

+

MDR: multidrug resistant; EA: ethyl acetate; ‘+’: present; ‘–’: absent.

Implicitly, H. antidysenterica was identified as the most effective plant with antibacterial efficacy.

analysis of phytoextracts demonstrated the presence of several secondary metabolites. For example, ethanol leaf extract of C. fistula and methanol leaf extract of H. antidysenterica contained all of 8 phytochemicals that we tested for. Ethanol and methanol extracts of C. fistula and H. antidysenterica had the largest zones of inhibition of all the solvents used on these plants; these results were corroborated by the recorded MIC and MBC values. The E. coli strain was the most susceptible to all the plant extracts, whereas the V. cholerae was the least susceptible strain. January 2015, Vol.13, No.1

4 Discussion Most published works on the use of plant extracts describe in vitro antibacterial effects against standard strains of bacteria from culture collection centres[13–15]. The present work records in vitro effects of plant extracts on pathogenic strains of bacteria isolated from clinical samples from

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difficult cases of diarrhoea. The resistance of these strains to commonly used antibiotics can result in potentially life-threatening infections when managed using traditional treatment approaches. Moreover, a major concern of enteropathogens stems from their ability to enter body’s vital organs, as well as transfer to other patients involved with under-5 children in the community setting[1]. Thus, there is a need to find new compounds to control MDR enteropathogens. These may include complementary and integrative phytomedicines, along with mainstream medicines or antibiotics in order to effectively combat child mortality. C. fistula is a rich source of secondary metabolites. Notably its phenolic compounds, tannins, flavonoids and glycosides, have a large number of pharmacological qualities, including antibacterial, antidiabetic, antifertility, anti-inflammatory, antioxidant, hepatoprotective, antitumor and antifungal activities[16]. The methanol leaf extract of C. fistula has been reported to be active against 2 Gram-positive and 2 GN bacteria[27]. Another study found that petroleum ether, chloroform, ethanol, methanol and water leaf extracts of C. fistula also inhibited the growth of E. coli, S. typhimurium, S. sonnei, Bacillus subtilis, B. licheniformis, S. aureus and S. epidermidis. The average MIC values of these extracts ranged between 94 and 1 500 μg/mL. Phytochemicals, alkaloids, flavonoids, carbohydrates, glycosides, protein and amino acids, saponins and triterpenoids were reported to be present in these extracts[28]. In a South-Indian report, antibacterial efficacy of fistulin, a plant protease inhibitor isolated from the leaves of C. fistula, exhibited significant antibacterial action against S. aureus, E. coli, B. subtilis and K. pneumonia; the efficacy was comparable to the standard drug, streptomycin sulphate[29]. Antibacterial efficacy of 16 medicinal plants from Bangladesh, including H. antidysenterica was reported against 7 enteric bacteria; the preparation of crude medicines from plant extracts was also reported, which was highly effective in controlling the enteric bacteria than the rest 15 plants used[30]. Further, the enterohaemorrhagic E. coli (EHEC) strain O157:H7 was also reported to be controlled by H. antidysenterica from Thailand[31]. The phyto-chemical 2,6-diisopropylphenol (propofol) was prepared by coupling 9-hydroxy-11-Z-octadecenoic acid, isolated from seed oil of H. antidysenterica with the C1-α-hydroxy function group of 2,6-diisopropylphenol. This compound was found to have antibacterial and anticancer properties[32]. One Indian study reported 3 new antifungal glycoside compounds extracted from the roots of T. alata[33]. Another study reported the antifungal activity of aqueous, ethanol and ethyl acetate leaf extracts of T. alata against Aspergillus flavus, A. niger, Alternaria brassicicola, A. alternata and Helminthosporium tetramera[34]. In another Indian study, antimicrobial activity of methanol, Journal of Integrative Medicine

ethanol, acetone, and aqueous (both hot and cold) extracts of T. arjuna leaves and bark against S. aureus, Acinetobacter sp., P. mirabilis, E. coli, P. aeruginosa and the fungus C. albicans, were established. Of these extracts, the acetone leaf extract was most effective. In the same study, the solvent extracts of T. arjuna bark had similar growth inhibition in all tested GN bacteria, except against P. aeruginosa, where the efficacy was less[21]. Phytochemical screening of bark extracts of T. arjuna revealed the presence of phenols, flavonoids, tannin, saponin, alkaloids, glycosides, phytosterols and carbohydrate. Methanol and ethanol extracts showed antibacterial activity at higher concentrations against the pathogens S. aureus, B. subtilis, B. cereus, E. coli, S. typhi, V. cholerae and K. pneumoniae than the other solvent extracts[35]. The anti-ulcer, anti-diarrheal, antidiabetic, antioxidant, antihelminthic, and hepatoprotective activities of T. arjuna were attributed to the presence of phytocompounds, peruloside, paederosidic acid, phenolic compounds, alkaloid, volatile oil, sitosterols, stigmasterol, campesterol, ellagic acid, lignans, iridoids, methylinedioxy compound, tannins, triterpenoids, urosil acid, and epifriedelinol[22]. Antibacterial activity of this plant against Helicobacter pylori has also been shown[36]. Furthermore, with bioassay-guided fractionations, antimicrobial effectiveness of n-hexane, chloroform and ethyl acetate fractions of methanol extracts of T. arjuna against Bacillus cereus, B. megaterium, B. subtilis, S. aureus, Sarcina lutea, E. coli, P. aeruginosa, S. paratyphi, S. typhi, Shigella boydii, S. dysenteriae, Vibrio mimicus, Vibrio parahemolyticus, as well as pathogenic fungi, C. albicans, A. niger and Sacharomyces cerevisiae were reported[37]. Ethanol and aqueous extracts of 10 Indian medicinal plants were tested for their antibacterial properties against S. sonnei, S. boydi, S. flexeneri, S. dysenteriae and E. coli; aqueous extract of Alium sativum was effective against S. dysenteriae, S. boydii, and E. coli. The ethanol extract of the rind of Garcinia mangostana inhibited growth of S. sonnei[38]. Antibacterial activity, forming an inhibition zone size > 13 mm against S. aureus, V. cholerae and E. coli, was shown in aqueous and ethanolic extracts of M. oleifera[39]. The ethanol extract of Punica granatum peel exhibited high antibacterial activity against 16 different serotypes of Salmonella species, and MIC values ranged between 62.5–1 000 µg/mL[40]. Cyclohexane, dichloromethane, ethyl acetate and butanol extracts of Chromolaena odorata L. leaf tissue had significant antibacterial activity against 4 clinical diarrheal strains, K. oxytoca, S. enterica, S. sonnei and V. cholerae, with MIC values at 0.156 and 1.25 mg/mL[41]. 5 Conclusion The 5 plants, with ethnomedicinal importance in India,

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used in this study, exhibited in vitro control over a cohort of 8 clinically isolated enteropathogenic bacterial strains. All these plants have potential for synergistic/integrative drug development for use in mainstream medicine. A formulation for use can be determined after isolation and characterization of the bioactive compounds responsible for the antibiotic activity of these plants. Obvious host toxicity testing is a critical step in assessing the utility of each candidate herb. Nevertheless, these 5 plants are listed in Indian pharmacopeia as edible medicinal plants. Development of these complementary treatments is important in the context of Asian countries, where infant/child mortality is much greater than in more developed nations. Obviously, cost-effective drugs are always sought after in resource-limited areas.

6

7

8

9

6 Acknowledgements This work is a part of PhD thesis in Microbiology of Utkal University of Shakti Rath, a Senior Research Fellow in a project from CSIR, New Delhi [No. 21 (0859)/11/EMRII] awarded to RN Padhy. Dr. R Sarangi, Associate Professor, Department of Paediatrics helped in data interpretation. We are grateful to Prof. Dr. MR Nayak, President and Sri G Kar, Managing Member, IMS and Sum Hospital for extended facilities. We are also thankful to Principal, BJB Autonomous College, Bhubaneswar, for encouragements.

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11

7 Competing interests

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January 2015, Vol.13, No.1