Patil & Gaikwad, IJPSR, 2011; Vol.

2(3): 614-619 ISSN: 0975-8232

IJPSR (2011), Vol. 2, Issue 3 (Research Article)

Received on 28 October, 2010; received in revised form 21 January, 2011; accepted 11 February, 2011

PHYTOCHEMICAL EVALUATION AND BACTERICIDAL POTENTIAL OF TERMINALIA ARJUNA

STEM BARK

Udaysing Hari Patil*1 and Dattatraya K. Gaikwad2

Department of Botany1, Bhogawati Mahavidyalaya Kurukali, Tal- Karveer, Dist-Kolhapur,

Maharashtra, India
Department of Botany2, Shivaji University Kolhapur, Maharashtra, India

ABSTRACT

Antibacterial activity of methanolic extract of Terminalia

Keywords:
arjuna stem bark samples (Apical bark, middle bark and
Antibacterial activities, Mature inner bark) was studied with respect to different
Terminalia arjuna,
pathogenic bacteria species Bacillus subtilis, Staphylococcus
Phytochemical analysis,
Extract yield,
aureus, Escherichia coli, Pseudomonas aeruginosa,
Secondary metabolites Klebsiella pneumoneae, Salmonella typhii, Proteus mirabilis
and Micrococcus sp. The methanolic extract of apical bark
was effective than the middle bark and mature bark in
Correspondence to Author: inhibiting the growth of all bacteria. The bacterium
Staphylococcus aureus was most sensitive among all the
Dr. Udaysing Hari Patil
bacterial species studied. Preliminary phytochemical
Assistant Professor, Department of analysis revealed the presence of alkaloids, glycosides,
Botany, Bhogawati Mahavidyalaya, flavonoids, flavanols, phenols, saponins and terpenoids.
Kurukali, Tal- Karveer, Dist-
Kolhapur, Maharashtra, India The concentrations secondary metabolites was found
higher in the apical stem bark than the middle and mature
stem bark. The percent extract yield was maximum in apical
stem bark.

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INTRODUCTION: The pathogenic microbial MATERIALS AND METHODS:

infectious agents developed chemotherapeutic and Collection and processing of the plant material:
antibiotic resistance has lead to screening of Different bark samples (Apical bark, middle bark
several medicinal plants for their potential and mature inner bark) of Terminalia arjuna were
antimicrobial activity 1. Higher plants as a source of collected from the hilly regions of Kolhapur district.
medicinal compounds plays a dominant role in The bark was collected in the month of May 2009.
human health 2. Various studies have been carried The bark samples were cut into pieces, sun-dried
out to extract the various natural products for then oven dried at 60oC. Dried bark samples were
screening antimicrobial activity 3, 4, 5. Plant based ground into powder and stored in an air tight
anti- microbials have enormous therapeutic plastic container.
potential. They are effective in the treatment of
infectious disease, simultaneously mitigating many Microorganisms: Bacillus subtilis, Staphylococcus
of the side effects that are associated with the aureus, Escherichia coli, Pseudomonas aeruginosa,
synthetic antimicrobials. Medicinal plants Klebsciella pneumoneae, Salmonella typhii, Proteus
represent a rich source of antimicrobial agent 6. It mirabilis and Micrococcus sp. were used for testing
has been estimated by WHO that 80% of the antibacterial activity of bark extracts. The test
people living in the developing countries rely upon organisms used in this study were obtained from
the traditional health practices for their primary the department of Microbiology, Shivaji University,
health care needs 7. Kolahapur, Maharashtra, India. The bacterial
strains were cultured on nutrient agar slants. The
Terminalia arjuna belongs to the family cultures were maintained by subculturing
combretaceae. T. arjuna is distributed throughout periodically and preserved at 4oC until further use.
India, Burma and Sri Lanka and mainly grows along
the banks of the river and streams 8. Arjuna is a Preparation of the extract: Oven dried 10g of
known potent cardiotonic since ancient times 9. powdered bark material was weighed accurately
Bark powder boiled with water and is inhaled to and placed in soxhlet extraction chamber which
cure headache and to kill worms in the teeth 10. was suspended above the flask containing 100mL
According to Kumar and Jain 11, people living in the of 80% methanol and below a condenser. The flask
South Surguja district of Madhya Pradesh uses the was heated and the methanol evaporated and
bark of T. arjuna in the treatment of fever and high moved into the condenser where it was converted
blood pressure. People living in the Malkangiri into a liquid that trickled into the extraction
district of Orissa, chew the fresh bark of Terminalia chamber containing the plant material. The
arjuna and the juice is used as antacid 12. Decoction extraction chamber was designed so that when the
of the bark is used as ulcer wash, while bark ash is solvent surrounding the sample exceeded at
used in the treatment of the snakebite and certain level it overflowed and trickled back down
scorpion sting 9. Paste of bark in water is applied into the boiling flask. At the end of the extraction
externally on the site of fractures and helps in the process, the flask containing the methanol extract
early healing. Taking into account of this wide use was removed and methanol was evaporated by
bark of Terminalia arjuna, present study is aimed using rotary evaporator. The weight of the residual
to evaluate the antimicrobial potential of its bark extract was measured and percent yield was
samples against the different human pathogenic calculated. The residue of the extract was
bacteria. dissolved in 25ml of pure methanol and stored in
air tight glass vials at 40C until further use 13.

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Extract yield % = W1 X 100 Determination of antibacterial activity by agar

W2 well diffusion method: Agar well diffusion method
Where; W1= Net wt of powder in grams after was employed to determine antibacterial activity
extraction; W2= total wt of wood powder in grams 14
. Well of 10mm diameter was prepared with
taken for extraction. sterilized cork-borer. Standard antibiotic
Chloramphenicol at 50µg/ml was served as positive
Qualitative screening of phytochemicals: Different
control and Methanol as negative control. The
extracts were screened for the presence of
plates inoculated with different bacterial species
alkaloids, glycosides, flavonoids, flavanols, phenols,
were incubated at 37oC in incubator for 24h and
saponins and terpenoids by using standard
the zone of inhibition was measured (Diameter in
protocols 15, 16.
mm). All of the experiments were performed in
Preparation of the media: Accurately weighed 28g triplicate. The results are reported as the average
of nutrient agar (Himedia) was dissolved in the of 3 experiments.
1000ml of distilled water. The medium was RESULTS: Preliminary phytochemical screening
sterilized under 15lb pressure for 15 minutes in an (Table 1) of methanolic extract of bark revealed the
autoclave. 30ml of this sterilized semisolid nutrient presence of some secondary metabolite alkaloids,
agar medium was poured in pre-sterilized 90mm glycosides, flavonoids, flavanols, phenols, saponins
glass petriplates under aseptic conditions in and terpenoids.
laminar flow. The plates were allowed to cool at
room temperature to solidify the medium.
TABLE 3: ANTIBACTERIAL ACTIVITY OF STEM BARK OF TERMINALIA ARJUNA
Minimum inhibitory concentration Zone of inhibition (Diameter in ‘mm’)*
Microorganisms Bark sample 25µL 50 µL 100 µL 200 µL 300 µL M C (50µg/ml)
1 0.00 1.70 ± 0.43 3.63 ± 0.50 5. 67 ± 0.00 8.67 ± 0.52 0.00
Bacillus subtilis 2 0.00 1.00 ± 0.57 3.37 ± 0.52 5. 00± 0.57 7.50 ± 0.55 0.00 21.50 ± 1.05
3 0.00 0.00 1.50 ± 0.51 3.50 ± 0.55 6.67 ± 0.52 0.00
1 0.00 2.39 ± 0.21 5.50 ± 0.22 8.33 ± 0.42 10.00 ± 0.63 0.00
Staphylococcus aureus 2 0.00 1.76 ± 0.26 5.5 ± 0.72 8.00 ± 0.51 10.50 ±0.67 0.00
11.17 ± 0.98
3 0.00 1.41 ± 0.42 5.33 ± 0.45 8.83 ± 0.45 10.33 ± 0.53 0.00
1 0.00 1.57 ± 0.11 3.33 ± 0.52 6.67 ± 0.58 8.67 ± 0.62 0.00
Escherichia coli 2 0.00 1.33 ± 0.19 3.17 ± 0.41 6.00 ± 0.63 8.33 ± 0.63 0.00
16.50 ± 0.55
3 0.00 1.11 ± 0.23 3.17 ± 0.41 5.50 ± 0.84 8.00 ± 0.52 0.00
1 0.00 2.00 ± 0.30 6.33 ± 0.52 8.67 ± 0.82 11.00 ± 0.63 0.00
Pseudomonas aeruginosa 2 0.00 1.87 ± 0.17 4.67 ± 0.82 8.00 ± 0.61 10.33 ±0.77 0.00
14.33 ± 0.5
3 0.00 1.12 ± 0.88 4.50 ± 0.55 7.83 ± 0.73 11.00 ± 0.63 0.00
1 0.00 2.10 ± 0.19 4.83 ± 0.75 6.83 ± 0.52 10.67 ± 0.82 0.00
Salmonella typhii 2 0.00 1.53 ± 0.00 3.83 ± 0.75 6.33 ± 0.41 9.33 ± 0.82 0.00
16.67 ± 1.21
3 0.00 1.13 ± 0.31 3.17 ± 0.75 6.17 ± 0.77 9.17 ± 0.69 0.00
1 0.00 2.58 ± 0.43 3.87 ± 0.02 4.53 ± 0.88 7.29 ± 0.31 0.00
Klebsiella pneumoneae 2 0.00 1.76 ± 0.27 2.70 ± 0.21 3.17 ± 0.65 5.83 ± 0.38 0.00 14.85± 1.17
3 0.00 1.60 ± 0.69 1.97 ± 0.36 2.24 ± 0.18 4.73 ± 0.53 0.00
1 0.00 1.89 ± 0.25 2.31 ± 0.67 5.41 ± 0.91 8.19 ± 0.29 0.00
Proteus mirabilis 2 0.00 1.53 ± 0.36 1.88 ± 0.74 3.69 ± 0.15 6.33 ± 0.13 0.00 13.70 ±0.49
3 0.00 1.02 ± 0.63 1.37 ± 0.41 2.47 ± 0.61 5.77 ± 0.86 0.00

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1 0.00 2.17 ± 0.55 3.89 ± 0.75 5.82 ± 0.17 7.93 ± 0.59 0.00
Micrococcus sp 2 0.00 1.70 ± 0.69 2.44 ± 0.35 4.73 ± 0.10 6.66 ± 0.26 0.00
16.63 ± 0.23
3 0.00 1.00 ± 0.23 2.29 ± 0.76 4.00 ± 0.07 5.49 ± 0.58 0.00
1: Apical Bark, 2: Middle Bark and 3: Mature inner Bark; C: Chloramphenicol M: Methanol; *: Agar well diffusion method; Values are mean ±
SD of three replicates
The concentration of phytochemicals was found Antibacterial activity of stem bark extract of
higher in the apical stem bark than the middle bark Terminalia arjuna against different bacterial
and mature bark. The apical stem bark shown pathogens is presented in the Table 3. From Table
higher percent extract yield than the middle and 3 it is clear that all the bacterial pathogens were
mature bark on main trunk (Table 2). inhibited at minimum inhibitory concentration of
50µL of all the bark extracts (Apical, middle and
TABLE 2: PERCENT EXTRACT YIELD mature bark extracts.). The inhibitory effect was
Plant name Bark sample Summer
increased with dose dependant manner and at a
Apical bark 26.11%
Terminalia maximum concentration of 300µL all bacteria were
arjuna Middle bark 25.87%
Mature inner bark 24.29% highly inhibited. The apical bark showed maximum
inhibitory effect than the middle and mature
bacteria.
TABLE 2: PHYTOCHEMICAL ANALYSIS OF METHANOLIC EXTRACT OF BARK OF TERMINALIA ARJUNA
Sample Phenols Flavones Flavonoids Tannin Terpenoids Saponin Alkaloids Cardiac glycosides
1 +++ +++ +++ +++ +++ +++ +++ +++
2 ++ ++ ++ ++ ++ ++ ++ ++
3 + + + + + + + +
1-Apical bark; 2-Middle bark and 3- Mature inner bark; +++: Present in high concentration, ++: Present in moderate concentration and +:
Present in low concentration

At maximum concentration of 300µL, the bacteria exhibited the antimicrobial activities against
Staphylococcus aureus and Pseudomonas bacteria S. typhii, K. pneumonae, P, aeruginosa,
aeruginosa were highly inhibited and showed and B. subtilis and four yeast species Candida
maximum inhibition zones in the range 10mm and albicans, Candida tropicalis, Candida parapsilosis
11mm respectively. The bacteria Escherichia coli and Cryptococcus neoformans respectively. Many
and Salmonella typhii showed inhibition zones in xanthons and anthraquinons have shown this type
the range of 8mm and 9.50mm respectively while of activity 20, 21. Eight flavonoids betulin, betulinic
the inhibition zone bacterium Bacillus subtilis was acid, oleanolic acid, quercetin, (-) epicatechin, gallic
in the range of 7mm. The three bacteria namely acid, and β-systosterol isolated from methanol
Klebsiella pneumoneae, Proteus mirabilis and fraction of Brysoniama crassifolia. These
Micrococcus sp were found resistant with minimum compounds were found active against many
inhibition zone in the range 5.5mm. bacteria including S. aureus, B. subtilis, K.
pneumonae and P. aureginosa 22. The bark extract
DISCUSSION: Flavonoids, tannins, alkaloid, and was found to be containing tannin glycosides,
other secondary metabolite serve as defense alkaloids, steroids and Flavonoids which are
mechanism against predations by many micro- biologically active 23. Phenolics and polyphenols
organisms, insect and herbivores 17, 18. Tamokou et present in the plants are known to be toxic to the
al., 19 isolated xanthones, physcion, friedelin and microorganism 24. Tannin from Dichrostachys
friedelanol, of these, xanthones and physcion cinerea root bark possesses antibacterial activities

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against S. aureus, E. coli and P. aeruginosa 25. 5. Cowan MM: Plant products as antimicrobial agents. Clinic
Microbiol. Rev., 1999; 12: 564-582.
Flavonoids have been reported to have both 6. Mahesh B and Satish S: Antimicrobial activity of some
antibacterial and antifungal activities 26. In our important medicinal plants against plant and human
study all the three bark samples revealed the pathogens. World J. Agri. Sci., 2008; 4(S): 839-843.
7. Austin DF and Bourne GR: In vitro cell development. Biol
presence of secondary metabolite. The high Plant., 1992; 33:111-113.
inhibitory potential of methanolic extract might be 8. Kirtikar KR and Basu BD: Indian medicinal plants Vol.2,
due to the high solubility of the phytoconstituents Jayad press Dehli. 1975; Pp 236.
9. Yesodharan K and Sujana KA: Ethnomedicinal knowledge
in the polar organic solvent like methanol. among Malamalasar tribe of Parambikulam wildlife
Presence of these phytoconstituents in the stem sanctuary, Kerala. Indian J. of Traditional Knowledge,
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10. Muthu C Ayyanar Raja N and Ignacimuthu S: Medicinal
of Terminalia arjuna and supports the claim of the plants used by traditional healers in Kancheepuram
traditional users. District of Tamil Nadu, India. J of ethnobilology and
Ethnomedicine, 2006; 2:43.
CONCLUSION: The reasons for high inhibitory 11. Kumar V and Jain SK: A contribution to Ethnobotany of
Surguja district in Madhya Pradesh, India. Ethnobotany,
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the apical, middle and mature stem bark that the 13. Harborne JB: Phytochemicals methods. Chapman and Hall,
active principle present in the apical bark might be New York. 1973; Pp95-120.
absent in the mature bark and if present may not 14. Perz C Paul M and Bazerque P: Antibiotic assay by agar-
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potential among the three bark sample. The high Pharmocognostic studies on Merugan kizhangu. Bull. Med.
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17. Lutterodt GD Ismail A Basheer RH Baharudin HM:
might be due to presence or synthesis of some new Antimicrobial activity of Psidium guajava extracts as one
active phytochemical. Thus, it is of prime important mechanism of its antidiarrhoeal action. Malaysian Rev. J.
to know the biochemistry of apical bark in order to Med. Sci. 1999; 6(2):17-20.
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screen the new pharmacological active principles. Microbiol. Rev., 1999; 12(4): 564-582.
19. Tamokou JDD Michel FT Wabo HK Kuiate JR and Tane P:
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