International Journal of PharmTech Research

CODEN (USA): IJPRIF, ISSN: 0974-4304

Vol.8, No.2, pp 244-249, 2015

Antimicrobial screening and phytochemical analysis of Aegle

marmelos against enteric pathogens
R. Selvaraj*, S. Jansi Rani, N. Mahesh Kumar and A. Natarajan
Centre for Laboratory Animal Technology and Research,
Sathyabama University, Chennai, India

Abstract: Aqueous and solvent extracts of leaves collected from Aegle marmelos plant leaves
were screened against enteric pathogens such as Escherichia coli, Salmonella spp., and
Shigella spp. This study initiated with isolation of test pathogens from enteric patients. Three
pathogens were isolated from 50 patients and used as test pathogens. Ethyl acetate and
acetone extract of Aegle marmelos showed antibacterial activity against test pathogens.
Phytochemical study of Aegle marmelos shows presence of steroid, carbohydrate, alkaloid,
phenolic compounds, saponins, xanthoprotein, tannins and flavonoids in ethyl acetate
fraction. The study proves that compounds from Aegle marmelos will be a good source for
diarrhea causing organisms.
Keywords: Antimicrobial, Aegle mermelos, Diarrhea.

Introduction

Diarrhoea is the third leading cause of morbidity and mortality in developing countries especially
among young children. Diarrhoeal disease is a major contributory factor to malnutrition. Recurrent diarrhea
coupled with inadequate feeding results in impaired body defense mechanisms. For infants and children in
India, diarrhoea is one of the leading causes of death. Sixty-eight percentage of the diarrheal disease occurs in
young children and it accounts for 17 % of death in children’s below 5 years. In HIV infected persons diarrhoea
can be prolonged severe and life threatening1. Diarrhoea is mainly transmitted through faecal by oral route. It is
caused by a wide variety of bacterial, viral and protozoan pathogens excreted in the faeces of humans and
animals. Escherichia coli, Salmonella spp., Shigella spp., Campylobacter jejuni, Vibrio cholerae, Rotavirus,
Norovirus, Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica2 are the major causative agent of
diarrhoea. Majority of diarrhoel disease is caused by bacterial pathogens in developing countries while virus
and protozoa tend to cause diarrhoea in developed countries.

In many parts of the world there is a rich tradition in the use of herbal medicine for the treatment of
infectious diseases. Medicinal plants have been used for centuries as remedies for human diseases because they
contain components of therapeutic value. Recently, the acceptance of traditional medicine as an alternative form
of health care and the development of microbial resistance to the available antibiotics has led authors to
investigate the antimicrobial activity of medicinal plants3, 4, 5, 6.

Microorganism like E.coli, Salmonella, Shigella, Camphylobacter, Yersinia, Aeromonas, Plesimonas

etc., are involved in the process of diarrhea. Several plant species have been reported in controlling various
human pathogens7.

Aegle marmelos is a medicinal plant which is a fairly large, deciduous and glabrous tree with auxillary
spines and usually trifoliate leaves belonging to the family, Rutaceae8. Aegle marmelos is commonly known as
R. Selvaraj et al /Int.J. PharmTech Res. 2015,8(2),pp 244-249. 245

vilvam in Tamil, bael in Hindi, sripal or bilwa in Sanskrit and bael tree in English. It is claimed to be useful in
treating pain, fever, inflammation, respiratory disorders, cardiac disorders, dysentery and diarrhoea.The leaves
extract of Aegle marmelos consist of tannins, skimmianin, essential oil (mainly caryophyllene, cineole, citral,
citronellal, d-limonene and eugenol), sterols and/or triterpenoids, including lupeol. Essential oil obtained from
leaves was found to possess broad spectrum anti bacterial and anti fungal activity9. Based on enteric diseases
and importance of medicinal plant, the study was aimed to test the antimicrobial potentials of Aegle marmelos
against enteric pathogens.

Materials and Methods

Collection of sample

Retrospective study was undertaken for a period of 6 months. Total of 50 cases, suspected with acute
gastroenteritis admitted in KMC hospital, Tiruchirappalli, Tamil Nadu was subjected to microbial investigation
at Department of Microbiology. Stool sample was collected from all the patients and transferred to laboratory
with help of transferred medium.

Microscopic examination

The stool samples were examined under microscope for the presence of parasites by iodine wet mount
method. The presence of protozoa and helminthes were identified by their morphology.

Isolation of Microorganisms

Escherichia coli, Salmonella spp., and Shigella spp.

Based upon the suspecting microorganisms, the mediums were prepared. The sample from the
transferred medium was inoculated into Hektoen enteric agar, Xylose-lysine deoxycholate agar and Salmonella
Shigella agar for isolation of E.coli, Salmonella, and Shigella bacteria. To differentiate Salmonella spp., from
Shigella spp., the sample was inoculated into Rajhans medium. All the plates were incubated at 37 °C for 24–
48hours.

Vibrio species and Aeromonas species

The sample also inoculated into Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar for the isolation of
Vibrio species, Ampicillin blood agar for isolation of Aeromonas species. The inoculated plates were incubated
at 37 °C for 24–48hours.

Campylobacter spp. and Yersinia enterocolitica

The stool sample was inoculated on Campy blood agar plates and incubated under microaerophilic
condition for 2 days. Colony formation in the medium was subjected to biochemical tests to confirm
Camplylobacter spp., The Stool specimen was directly streaked on Yersinia selective medium and incubated at
37 °C for 24 h. The plates were observed for dark pink colonies after overnight incubation for the isolation of
Yersinina enterocolitica

Antibiotic sensitivity test

All the isolated pathogens were analysed for antibiotic sensitivity test by Kirby Bauer method. About
18 h old bacterial culture was prepared and inoculated into Mueller Hinton Agar (MHA) plates. Standard
antibiotics disc were placed on MHA plates inoculated with test bacterial strains. All the plates were incubated
at 37 °C for 24 h.

Collection of plant leaves

The plant Aegle marmelos leaves were collected from in around Tiruchirrapalli. The freshly collected
leaves were washed and dried in shade at room temperature for 10–15 days. The dried leaves were used for
powdering by using mortar and pestle. The larger plant debris was removed and powdered leaves were used for
extraction for antimicrobial compounds.
R. Selvaraj et al /Int.J. PharmTech Res. 2015,8(2),pp 244-249. 246

Extraction of crude compounds

Aqueous extract

One gram of fresh leaves were taken and washed with sterile distilled water. The leaves were crushed
by using mortar and pestle. The crushed leaf paste was mixed with 20 ml of sterile distilled water in 50 ml
beaker. The aqueous leaf mixture was covered with aluminium foil and kept at room temperature for 24 hrs.

Solvent extract

Five different solvents such as methanol, chloroform, ethyl acetate, dichloromethane and acetone were
used for the extraction of antimicrobial compounds from leaf powder. One gram of dried plant powder was
taken in a 50 ml beaker and 20 ml of methanol was added into it. This content was mixed well and the beaker
was covered with aluminium foil and kept for extraction at room temperature for 24 h. The procedure was
adopted for remaining solvents.

Antibacterial activity of crude extracts

Aqueous extracts

The antimicrobial activity of mangrove aqueous extracts was studied by well diffusion method using
MHA plates. About 18 hours old bacterial culture was prepared and inoculated into MHA plates. 5 mm
diameter well was cut on plates. Each 10µl of aqueous plant extracts were added in wells using micropipette.
Ten micro liter sterile distilled water was used as a control well. All the plates were incubated at 37 °C for 24 h
and plates were observed for zone of inhibition6.

Solvent extracts

The antimicrobial activity of solvent extracts was studied by disc diffusion method using MHA plates.
About 18 h old bacterial cultures were inoculated into MHA plates. 0.25 mg of crude extracts were added into
sterile filter paper disc (5 mm diameter) and allowed to dry at room temperature for few minutes. Crude plant
extract impregnated discs were placed on MHA plates inoculated with test bacterial strains. Sterile empty disc
was used as a control. All the plates were incubated at 37 °C for 24 h. After incubation the plates were observed
for zone of inhibition.

Phytochemical study

Methanol extract was refluxed with 2N HCL in methanol. This concentration was saponified with 5 %
KOH in ethanol. Solvent was removed by evaporation under reduced pressure and diluted with water. Then the
mixture was extracted several times with chloroform. The extracted compound was dissolved in chloroform and
analyzed for the presence of flavanoids, alkaloids, terprnoids, saponins, tannins, amino acids, anthraquinone,
steroids, glycosides and reducing sugar.

Result and Discussion

Microscopic examination and gross examination of stool specimen revealed, absence of adult worms in
all the 50 samples and 14 % of the sample showed positive to Entamoeba cyst. The prevalence of diarrhea with
reference to age showed that bacterial incidence was higher than any other microbial etiology. Incident rate of
diarrhea is higher among children who are 2–5 years of age (Table 1).

Table 1: Incidence of diarrhoea with reference to age

No. of Microbial etiology

Age
samples Bacteria Protozoans Nematodes Combination
0–1 year 13 13 Nil Nil Nil
2–5 years 17 14 3 Nil Nil
6–10 years 10 5 2 Nil 1
11–15 years 10 4 3 Nil 1
R. Selvaraj et al /Int.J. PharmTech Res. 2015,8(2),pp 244-249. 247

Isolation of pathogens

Totally three genus were isolated from stool samples. All the test pathogens were identified by cultural
characteristics and biochemical analysis and confirmed by Bergey's manual of systematic bacteriology.

Selective and differential culturing results showed that 50 % of the infection was due to E.coli followed
by Shigella 24 % and Salmonella 26 %. This result correlates with study of Paniagua et al. 1997 in which 38 %
of the diarrhoel episode is caused by E.coli10. The isolated E. coli, Salmonella spp., and Shigella spp., used as
test pathogens.

Antibiotic sensitivity test

Antibiotic sensitivity pattern of enteric isolates shows an alarming rate of increasing resistant properties
of the isolates. This result highly correlates with the result of Agarwal et al.11 The sensitivity pattern of isolated
test pathogens was represented in Table 2.

Table 2: Antibiotic sensitivity assay of enteric isolates

Quantity Percentage of resistance

S.No. Antibiotics
(mcg) E.coli Salmonella spp. Shigella spp.
1 Amikacin 30 S S S
3 Azithromycin 30 I S S
3 Vancomycin 30 S S S
4 Chloramphenicol 30 R R R
5 Amoxyclav 30 R I S
6 Kanamycin 30 R R R
7 Erythromycin 30 R I S
8 Tetracycline 30 R R R
9 Sreptomycin 30 S R R
10 Ampicillin 30 I I R
R- Resistant; I-Intermediate; S-Sensitive

Antimicrobial activity of Crude extract

Antimicrobial activity of plant Aegle marmelos aqueous extract does not showed any of activity against
test pathogens (E. coli, Salmonella spp., and Shigella spp.).

In this present study, solvents such as methanol, chloroform, ethyl acetate, dichloromethane and
acetone were tested for extraction of crude compound. Among the various solvents tested, the crude compounds
were extracted only in ethyl acetate and acetone but not in other solvents. Solvent extracts of Aegle marmelos
showed antibacterial activity with average zone of inhibition from 10 to 15 mm (Table 3). Aegle marmelos
grown as a tree has shown to have the antimicrobial activity due to the seed oil as their component12.

Table 3: Antibacterial activity of Aegle Marmelos crude extract

Zone of inhibition in mm
S.No. Extract
E. coli Salmonella spp. Shigella spp.
1 Aqueous – – –
2 Hexane – – –
4 Chloroform – – –
5 Ethyl acetate 15 12 11
6 Methanol – – –
7 Acetone 13 – 12

Phytochemical analysis of Aegle marmelos

Aegle marmelos showed positive results for the presence of steroid, carbohydrate, alkaloid, phenolic
compounds, saponins, xanthoprotein, tannins and flavonoids in ethyl acetate fraction. The other extract also
R. Selvaraj et al /Int.J. PharmTech Res. 2015,8(2),pp 244-249. 248

shows some of these compounds in acetone and chloroform (Table 4). Tannins, phenolics and flavonoids
contributed for the antibacterial property of the plant. This is similar to the effect observed by Randir et al.13

Table 4: Phytochemical analysis of Aegle marmelos

Reducing sugar

Xanthoproteins
Carbohydrates
Triterpenoids

Flavanoids
Alkoloids

Saponins
Extracts
Steriod

Tanins
Phenol
Hexane + – – – – + + – + +
Benzene – – – – – + + – + +
Chloroform – – – + – + + – + +
Ethyl – – – + + + + – + +
acetate
Methanol – – – + – + + – + +
Acetone + – – – – + + – + +
Aqueous + – – + – + + + + +
Water + – – + – + + – + +
+ –––> Present; – ––––> Absent

Conclusion
E.coli, Salmonella spp., and Shigella spp., were isolated in this study, among the three isolates E.coli
shows highest predominance (50 %). Aegle marmelos are traditional medicinal plants used for the treatment of
diarhoea. The findings of the present study conclude that Aegle marmelos plants will be a potential source for
production of bioactive compounds against enteric organisms like E. coli, Salmonella spp., and Shigella spp.
Further studies such as purification, chemical characterization and structure elucidation of active compounds
from the plant are in progress.

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