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Original Article
ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL ANALYSIS OF FRUIT EXTRACTS OF
TERMINALIA BELLERICA
P. NITHYA DEVI, S. KALEESWARI AND M.POONKOTHAI*
Department of Zoology, Faculty of Science, Avinashilingam Institute for Home Science and Higher education for Women, Coimbatore
641043, Tamil Nadu, India.
Email: poonkothaiadu@gmail.com
Received: 14 Apr 2014 Revised and Accepted: 21 May 2014
ABSTRACT
Objective: In current era, herbal products are measured to be the symbols of safety in comparison to the synthetic products that are regarded to be
hazardous to human life and environment. Although herbs had been priced for their therapeutic importance, their phytochemical and
pharmacological activities are conducted on different parts. With this, an attempt has been made to investigate the antimicrobial activity and
phytochemical analysis of Terminalia bellerica fruits.
Methods: The antimicrobial activity was evaluated using agar well diffusion method against the bacterial (Escherichia coli, Pseudomonas aeruginosa,
Klebsiella pneumonia, Shigella flexneri, and Salmonella typhi) and fungal (Aspergillus niger, Mucor species, Aspergillus fumigatus, Rhizopus species
and Aspergillus flavus) isolates using aqueous, petroleum ether and chloroform extracts of Terminalia bellerica fruits. Phytochemical and FT-IR
analysis was carried.
Results: It was observed that aqueous extract exhibited significant activity against the tested bacterial and fungal isolates, compared with
chloroform and petroleum ether extract respectively. Phytochemical analysis of Terminalia bellerica extracts showed the presence of secondary
metabolites like phenolics, alkaloids, flavonoids and tannins. The FT-IR analysis has revealed the presence of phenols, alcohol, amines and
carboxylic acid as functional groups in Terminalia bellerica.
Conclusion: From this study, it can be concluded that Terminalia bellerica reveal antimicrobial activity against various human pathogenic bacteria.
Keywords: Antibacterial, Antifungal, Terminalia bellerica, phytochemical and FT – IR analysis.
INTRODUCTION
Nature is and will still serve as mans primary source for the cure of
his ailments. However, the potential of higher plants as a source for
new drugs is still largely unexplored [1]. The traditional system of
using medicinal plants for curing many diseases dates back to the
age of Rig Veda. Many microbial diseases can be cured by medicinal
plants without any side effects and economical issues [2]. Multidrug
resistance towards antibiotics and their related effects has an added
effect to pursue the use of natural drugs [3]. Infection with various
microorganisms is one of the leading causes for a number of
diseases [4]. Infectious diseases are usually characterized by clear
symptoms, so it is likely that traditional healers have been able to
recognize such diseases and have developed effective therapies. In
recent past, there has been tremendous increase in the use of plant
based products in developing as well as developed countries resulting in
an exponential growth of herbal products globally. A variety of
phytochemicals are accumulated in plants accounting for their
constitutive antimicrobial activities. World Health Organisation (WHO)
noted that the majority of the world’s population depends on traditional
medicine for primary health care [5]. Terminalia bellerica
(Combretaceae), a large deciduous tree found throughout India has
enormous medicinal properties. The seed oil is used to cure skin
diseases, premature graying of hair and can be applied on painful
swollen parts. The fruits of bellerica can be used to treat cough, cold,
hoarseness of voice, asthma, arrest bleeding, boost hair growth, impart
black colour to hair, cure conjunctivitis, astringent and anti-diarrheal
agent. Fruit extract of T. bellerica produced fall in blood pressure of rats
at a concentration of 70 mg/kg body weight. The plant helps in loss of
appetite, piles, lowering cholesterol, blood pressure, boosts immunity
and prevents ageing. It also enhances the body resistance against
diseases. It is used as traditional medicine to get remedies from all the
above ailments by the local people of Coimbatore district [6].
Considering these facts, it is expected that the screening and
scientific evaluation of the fruits of bellerica may provide novel
antimicrobial compounds.
MATERIALS AND METHODS
All the chemicals and reagents used were from Hi- Media Pvt.
Limited, Bombay, India. Glass wares used were from Borosil.
Collection of fruit
The fruits of Terminalia bellerica was collected during January-
February 2013 in the areas in an around Coimbatore, Tamil Nadu.
The fruit was authenticated and a voucher specimen was kept in the
Department of Botany, Avinashilingam University for Women,
Coimbatore, Tamil Nadu, India. The fruits were washed thoroughly
under running tap water for 2 - 3 times to remove dirt and then
shade dried at room temperature for a week. The dry fruits, devoid
of seeds were ground into fine particles and kept in closed container
before being stored at room temperature until further used.
Preparation of fruit extract
Ten grams of the ground sample of Terminalia bellerica was weighed
and homogenized with 100 ml of petroleum ether, aqueous and
chloroform separately. The crude preparation was left overnight in the
shaker at room temperature and then centrifuged at 4000 rpm for 20
minutes. The supernatant containing the fruit extract was then
transferred to a pre-weighed beaker and the extract was concentrated
by evaporating the solvent at 60º C. For the preparation of aqueous
extract, 10 g of the sample was added with 100 ml of distilled water
and kept in a shaker at 90-120 rpm for 24 h at 30º C. The mixture was
boiled at 60º C for 3 h and concentrated to one fourth of the original
volume. The extracts were then concentrated to dryness under
vacuum and reduced pressure using rotary evaporator. Then the
crude extracts were dissolved in known volume of dimethyl
sulphoxide (DMSO) to obtain a final concentration of 20mg / 5 µl. The
aliquot was stored until it was used [7].
Microbial strains
The bacterial (Escherichia coli, Pseudomonas aeruginosa, Klebsiella
pneumonia, Shigella flexneri, and Salmonella typhi) and fungal
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 6, Issue 5, 2014
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Poonkothai et al.
Int J Pharm Pharm Sci, Vol 6, Issue 5, 639-642
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(Aspergillus niger, Mucor species, Aspergillus fumigatus, Rhizopus
species and Aspergillus flavus) isolates used in the present study
were the clinical isolates obtained from P.S.G. Hospitals, Coimbatore,
Tamil Nadu, India.
Culture media and inoculums preparation
Muller Hinton agar media / broth (Himedia, Mumbai, India) were
used as the media for the culturing of the bacterial strains. Loop full
of all the bacterial cultures were inoculated in the Muller Hinton
broth and incubated at 37ºC for 24 hrs. Rose Bengal
Chloramphenical agar/ broth (Himedia, Mumbai, India) were used
as the media for the culturing of fungal strains. Loop full of all the
fungal cultures were inoculated in the Rose Bengal Chloramphenical
broth and incubated at room temperature for 72 hrs.
Antimicrobial assay
Well diffusion method
The agar well diffusion method was employed for the determination
of antimicrobial activity of the extracts [8]. To brief, five wells were
made in Muller Hinton agar plates and Rose Bengal Chloramphenical
agar plates respectively using sterile cork borer (5 mm diameter).
50 µl of bacterial and fungal inoculum were swabbed on the above
plates with sterile swabs separately. 20 µl of each extract, control
(DMSO) and standard antibiotics (4 mg of Chloramphenical for
bacteria and nystatin for fungi) were filled in the respective wells
with the help of micropipette separately. The plates were then
incubated at 37ºC for 24 hours for bacteria and at room temperature
(25 - 30ºC) for five days for fungal isolates. The samples were tested
in triplicates and the diameter for the zone of inhibition was
measured as millimeter (mm) and the results were expressed as
mean ± standard deviation.
Phytochemical screening of the extracts
The extracts obtained from the fruits of Terminalia bellerica were
qualitatively tested to identify the presence of phytochemicals such
as alkaloids, phenols, amino acids, flavonoids, saponins, tannins,
quinones, carbohydrates, glycosides, steroids and terpenoids
according to the method proposed by [10].
FT – IR analysis
FT-IR (Fourier Transform Infrared) is a tool used for identifying the
types of chemical bonds (functional groups). The wavelength of light
absorbed is characteristic of the chemical bond which can be seen in
the annotated spectrum. By interpreting the infrared absorption
spectrum, the chemical bonds in a molecule can be determined. For
the FT-IR study dried powder of aqueous extract (10 mg) of
Terminalia bellerica fruits was taken in a mortar and pestle and
ground with 2.5 mg of dry potassium bromide (KBr). The powder so
obtained was filled in a 2 mm internal diameter micro-cup and
loaded onto FT- IR set at 26°C ± 1°C. The samples were scanned
using infrared in the range of 4000–400 cm
-1
using Fourier
Transform Infrared Spectrometer (Shimadzu, IR Affinity 1, Japan).
The spectral data obtained were compared with the reference chart
to identify the functional groups present in the sample [11].
Microdilution method
The minimum inhibitory concentration (MIC) was determined by
micro dilution method using serially diluted Terminalia bellerica
extracts according to the NCCLS protocol [9]. The aqueous extract
were diluted to get series of concentrations from 100mg/ml to
1.56mg/ml in in sterile Muller Hinton broth using 96 - well plates.
The microorganism suspension of 50µl was added to the broth
dilutions and was incubated for 18 hours at 37ºC. MIC of each
extract was taken as the lowest concentration that did not give any
visible bacterial growth.
RESULTS
The resu lts obtaine d are summ arized in Tabl e 1 indicating the
growt h inhibitio n produced by fruit extract of Term inalia
bellerica towards bact eria l and fungal isolates. The experimental
results obtai ned from the present st udy illustrates that the
aqueo us extract was found to be more effec tive to control the
bacterial a nd fungal growth w hen compared with chloroform
and petroleum ether extracts respectively. All the bacterial and
fungal isolates test ed sho wed significant activity against the
aqueo us extract and the zone o f inhibition ranged from
15 -23 mm. The chloroform extract of the fruits of Terminalia
bellerica showed moderate zone of inhi bition against the tested
bacterial and fungal isolates (9-15 mm). The petroleum ether
extracts of the fruits of Terminalia bellerica exhibited less zone
of inhibition (8 -13 mm) against the tested microorganisms. The
extracts exhibite d significant zone of inhibition when compared
with the tested standard antibiotics (Chlo ramphenicol and
nystatin) and no zone of inhibition was obs erve d i n ne gative
contr ol (DMSO). The highe st zone of inhibitio n was fou nd
against Klebs iella pneumon iae (23 mm) and Aspe rgillus fumigatus
(22 mm) with aqueous e xtract and least inhibition against
Salmo nella typh i (8 mm) and Aspergillus niger (9 mm) with
petro leum ether e xtract.
The minimum inhibitory concentration (MIC) of the extracts to inhibit
the microorganisms was determined using the microdilution method.
Since the aqueous extract showed the maximum zone of inhibition, the
MIC was determined only with this extract. Table 2 depicts the MIC
values of the extract against the tested bacterial and fungal isolates.
The aqueous extract could inhibit the growth of E. coli and A.
fumigatus at a minimum concentration of 6.25mg/ml when
compared with other microbial isolates (Table 2). The MIC values for
the standard antibiotics against the tested microbes were depicted
in Table 2. The results further validate the activity of aqueous
extracts against all the tested bacterial and fungal isolates.
The phytochemical analysis of the fruit extracts of Terminalia
bellerica was tabulated in table 3. It revealed the presence of
alkaloids, phenol, tannins and flavonoids.
Table 1: Antimicrobial activity of the fruit extracts of Terminalia bellerica
Microorganisms
Zone of inhibition in diameter (mm)
Petroleum ether
Chloroform
Aqueous
Positive control
Negative control
Escherichia coli
11.6±1.5
13.6±1.5
14.6±1.5
21.6±1.5
-
Pseudomonas aeruginosa
9.3±2.5
12.6±2.5
13.6±1.5
26±1.0
-
Klebsiella pneumoniae
12.6±1.5
14.3±2.0
22.6±2.5
18±2.5
-
Shigella flexneri
9.3±2.0
16
21.3±1.5
12±2.0
-
Salmonella
typhi
8±2.0
8.6±1.5
10±1.5
10.6±1.5
-
Aspergillus niger
9±1.0
11.3±2
17.6±1.5
20±1.0
-
Mucor
species
10.3±1.5
17
20.6±1.5
13.6±1.5
-
Aspergillus
fumigatus
13.3±1.5
14.6±1.5
19.3±1.5
23.3±1.5
-
Rhizopus
species
12±2.0
15±2.0
19.3±1.5
23.3±1.5
-
Aspergillus
flavus
10.3±1.5
10.3±1.5
20.3±2.5
24±1.0
-
Positive control – Chloramphenicol (Bacteria), Nystatin (Fungi), Negative control – DMSO
Poonkothai et al.
Int J Pharm Pharm Sci, Vol 6, Issue 5, 639-642
641
Table 2: Minimum Inhibitory Concentration of aqueous extract of Terminalia bellerica against bacterial and fungal isolates
Bacterial isolates
Concentration (mg/ml)
Standard Antibiotics
(mg/ml)
Escherichia coli
6.25
12.5
Pseudomonas aeruginosa
25
25
Klebsiella pneumoniae
50
100
Shigella flexneri
12.5
50
Salmonella
typhi
100
12.5
Aspergillus niger
50
25
Mucor
species
25
50
Aspergillus
fumigatus
6.25
12.5
Rhizopus
species
50
12.5
Aspergillus
flavus
12.5
100
Standard Antibiotics – Chloramphenicol (Bacteria), Nystatin (Fungi)
Table 3: Qualitative phytochemical analysis of fruit extract of Terminalia bellerica
Phytochemicals
Terminalia bellerica
extracts
Petroleum ether
Chloroform
Aqueous
ALKALOIDS
Dragendroff’s Reagent
+
+
+
Hager's test
-
-
-
Wagner’s Reagent
-
-
-
PHENOLS
Ferric chloride test
+
+
+
Lead acetate test
+
+
+
AMINO ACID
Ninhydrin test
-
-
-
FLAVONOIDS
Schinoda’s test
+
+
+
Lead acetate Test
+
+
+
SAPONINS
Froth test
-
-
-
TANNINS
Breamer's test
+
+
+
QUINONES
Borntrager's test
-
-
-
CARBOHYDRATES
Molish test
-
-
-
Fehling's test
-
-
-
GLYCOSIDES
Legal's
test
-
-
-
STERIODS/TERPENOIDS
Libermann
–
Burchardt test
-
-
-
The FT –IR spectrum of the aqueous extract of the fruits of Terminalia bellerica in the range of 400 – 4000 cm
-1
revealed the presence of many
functional groups. It exhibits the peak at 3950, 3749, 3417, 2924, 2854, 2376, 1720, 1627, 1442, 1381, 1226 and 1033 cm
-1
which indicates the
presence of –OH, -COOH, -NH and C=O groups respectively (Fig. 1).
Fig. 1: FT –IR spectrum of the fruit extract of Terminalia bellerica
Poonkothai et al.
Int J Pharm Pharm Sci, Vol 6, Issue 5, 639-642
642
DISCUSSION
Infectious diseases have become the major cause and serious
concern in public health issues. The occurrence of drug resistant
strains with less susceptibility to antibiotics due to mutation is
challenging amongst the researcher to invent newer drugs [12]. At
this scenario, evaluation of antimicrobial substances from various
sources of medicinal plants is considered to be a pivotal role. The
demonstration of activity against the test bacteria provides scientific
base for the local usage of this plant in the treatment of various
ailments. The fact that the extracts were active against bacterial and
fungal isolates tested may indicate a broad spectrum of activity. This
observation is very significant because of the possibility of
developing therapeutic substances that will be active against
multidrug-resistant organisms. The results of the study supports the
traditional application of the fruit extract of Terminalia bellerica and
suggests the presence of compounds with antimicrobial properties
that can be used as antimicrobial agents in novel drugs for the
treatment of microbial diseases [13].
The aqueous extract of the fruits of Terminalia bellerica confirmed
the antimicrobial effect on bacterial and fungal isolates, suggesting
that the phytochemicals present in the extract may deactivate
various cellular enzymes which play a vital role in metabolic
pathways of these microorganisms. It has also been found that the
phytochemicals may denature the proteins of the cells, which as a
result impairs normal cellular process.
A variety of phytochemicals present in the plant extracts are non-
nutrient compound possess biological activity that can be of valuable
therapeutic index. Different phytochemicals have been found to
possess a wide range of activities, which may help in protection
against chronic diseases [14]. The phytochemical screening of fruit
extract of Terminalia bellerica showed the presence of alkaloid, phenol,
tannins and flavonoids. The phytochemical alkaloid present in the fruit
extract might have inhibited the microorganism by impairing the
enzymes involved in energy production, interfering the integrity of cell
membrane and structural component synthesis. The growth of the
fungus might have been inhibited due to the presence of phenol which
might have induced the swelling, plasma seeping and leakage,
distortion, abnormal branching or fusion and wrinkling of hyphae.
Presence of tannins in the fruit extract of Terminalia bellerica might
have prevented the development of microorganisms by precipitating
the microbial protein and making nutritional proteins unavailable for
them [15]. It has also been reported that tannins have been found to
form irreversible complexes with proline rich proteins resulting in the
inhibition of cell protein synthesis [16]. The presence of characteristic
functional groups may be responsible for the medicinal properties of
Terminalia bellerica which contain high therapeutic content.
Determination of respective antimicrobial potential and toxicological
evaluation of these extracts with the view to formulate novel
chemotherapeutic agents to be used in future is worth mentioning.
CONCLUSION
The results of the study support the traditional application of the
fruit extracts which possess compounds with antimicrobial
properties that can be used in novel drugs for the treatment of
microbial diseases. Further pharmacological evaluations,
toxicological studies and possible isolation of the therapeutic
antimicrobial from this fruit are the future challenges.
ACKNOWLEDGEMENT
The authors wish to place their record of thanks to the authorities of
Avinashilingam Institute for Home Science and Higher education for
Women, Coimbatore, Tamil Nadu, India for providing infrastructure
facility.
REFERENCES
1. Oke OM, Hamburger PS. Screening of some Nigerian medicinal
plants for antioxidant activity using 22, diphenyl picryl –
hydrazyl radical. African Journal of Biomedical Research 2002;
5: 77- 79.
2. Nithya T, Kavitha PK, Gayathri U, Madhavan S, Venkatraman
BR. Antibacterial activity of Solanum trilobatum. J. Ecotoxicol
Environ.Monitoring 2004; 14 Suppl 3: 237 -239.
3. Kavitha D, Padma PR. A study of the antimicrobial effect of
Albizia amara leaf extracts. Advances in Plant Sciences 2011;
24 Suppl 1: 49-52.
4. Shivaii R, Naveet S, Jain AK. Antimicrobial activity of medicinal
plants against vaginal pathogens. Advances in plant sciences
2011; 24 Suppl 1: 53-55.
5. Prabuseenivasan S, Jayakumar M, Ignacimuthu S. In vitro
antibacterial activity of some plant essential oils. BMC
complementary and aternative medicine 2006; 6 Suppl 39;1-9.
6. Kirtikar KR, Basu BD. Indian medicinal plants. 2
nd
ed.
Dehradun; International Book distributors; 1999.
7. Beltrame FL, Pessini GL, Doro DL, Filho BPD, Bazotte RB, Cortez
DAG. Evaluation of antidiabetic and antibacterial activity of
Cissus sicyoides. Braz. Arch. of Bio. and Tech., 2002; 45 Suppl 1:
21 -25.
8. Bauer AW, Kirby WMM, Sherris JC, Truc M. Performance
standards for antimicrobial disc susceptibility. Am.J.Clin Pathol.
1996; 45: 493- 496.
9. NCCLS (National Committee for Clinical Laboratory Standards).
Methods for dilution Antimicrobial Susceptibility Tests for
Bacteria that grow aerobically, Approved Standard, Fifth
Edition, NCCLS document M7A5, Wayne, PA, USA, 2000.
10. Harborne JB. Phytochemical methods : A guide to modern
techniques of plant analysis. London: Chapman and Hall; 1998.
11. Naumann D, Helm D, Labischinski H, Giesbrecht P. The
characterization of microorganisms by Fourier-transform
infrared spectroscopy (FT-IR). In: Nelson WH, editor. In
Modern Techniques for Rapid Microbiological Analysis. New
York: VCH Publishers; 1991b.
12. Rani MS, Dayanand CD, Shetty J, Vegi PK, Kutty AVM. Evaluation
of antibacterial activity of Pongamia pinnata Linn on pathogens
of clinical isolates. American Journal of Phytomedicine and
Clinical Therapeutics. 2013;1 Suppl 8: 645-651.
13. Rana BK, Singh UP, Tanieja V. Antifungal activity and kinetics of
inhibition by essential oil isolated from leaves of Aegle
marmelos. J. Ethnopharmacol. 1997; 57: 29-34.
14. Gurib-Fakim A. Medicinal plants: Tradition of yesterday and
drugs of tomorrow. Review article. Mol.Aspects Med. 2006;
27(1): 93.
15. Hung JW, Chung WC. Management
of vegetable crops disease
with plant extracts. Advances in plant disease management.
2003; 37:153-163.
16. Hagerman AM, Butler IG. The specificity of pro-anthocyanidin-
protein interactions. J.Biol.Chem. 1981; 256: 4494 – 4497.
