ejbps, 2015, Volume 2, Issue 4, 511-518.
Sovan et al.
Research Article
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2349-8870
European
Journal of Biomedical
and Pharmaceutical ISSN
Sciences
European
Journal
of Biomedical
Volume: 2
AND
Issue: 4
511-518
Pharmaceutical sciences
Year: 2015
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PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL
ACTIVITIES OF LEAF EXTRACTS OF GUAVA(PSIDIUM GUAJAVA L.)
Ilma Siraj, Kamraj Singh and Sovan Bagchi*
Department of Biotechnology, Dolphin (P.G.) Institute of Biomedical and Natural Sciences,
Dehradun, Uttarakhand, India.
Article Received on 07/06/2015
Article Revised on 30/06/2015
Article Accepted on 23/07/2015
*Author for
ABSTRACT
Correspondence
The antimicrobial activity and phyto-chemicals from aqueous and
Sovan Bagchi
ethanol extracts of Psidium guajava leaves were evaluated. Aqueous
Department of
and ethanol extracts of Psidium guajava leaves show the presence of
Biotechnology, Dolphin
(P.G.) Institute of Biomedical
phytochemicals
like
alkaloids,
glycosides,
saponins,
phenolic
and Natural Sciences,
compounds, etc. The guava leaves exhibited anti bacterial activities in
Dehra Dun, Uttarakhand,
both the ethanol and aqueous extracts. The ethanol extracts of the
India.
guava leaf showed greater antibacterial activity as compared to
aqueous extracts. The present study was aimed to evaluate the role of
P. guajava extracts on microorganisms such as Klebsiella sp., Pseudomonas sp. and Proteus
sp. The antibacterial activity of these extracts give an idea about which extract is effective
against above mentioned bacteria after preliminary screening for antibacterial activity by well
diffusion method. Both the ethanol and aqueous extracts showed inhibitory action against the
growth of bacteria under study with varying degree of activity.
KEY WORDS: Plant extracts activity; Medicinal plants; Antimicrobial activity; Guava leaf.
INTRODUCTION
The guava (Psidium guajava L.) plant used in folk medicines and is believed to have active
components that help to treat various diseases. Many parts of the plant have been used in
traditional medicines to manage diseases like malaria, gastroenteritis, vomiting, diarrhea,
dysentery, wounds, ulcers, toothache, coughs, sore throat, inflamed gums, and a number of
other conditions.[1-3] This plant has also been used for the controlling of life style diseases
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such as diabetes, hypertension and obesity.[5-7] The aim of the present study was to evaluate
antibacterial efficacy of leaf extracts of guava using aqueous and organic (ethanol) solvents.
Guava (Psidium guajava) is an exotic fruit member of the family Myrtaceae. Guava, goiaba
or guayaba are some of the names given to the “apple of the tropics”, popular for its aroma
and flavor. Its place of origin is quite uncertain, extending in an area from southern Mexico
through Central and South America. Currently, its cultivation has been extended to many
tropical and subtropical parts of the world, where it also thrives well in the wild.[8-9]
The genus Psidium comprises approximately 150 species of small trees and shrubs in which
only 20 species produce edible fruits and the rest are wild with inferior quality of fruit.[10] The
most commonly cultivated species of Psidium is P. guajava L. which is the „common guava‟.
Other species are utilized for regulation of vigor, fruit quality improvement and resistance to
pest and disease.[10] Guava fruit today is considered minor in terms of commercial world
trade, but it is widely grown in the tropics, enriching the diet of hundreds of millions of
people in those areas of the world.
There are bioactive components in the guava leaf that can fight against pathogens, regulate
blood glucose levels, and can even aid in weight loss. The leaves of guava contain an
essential oil rich in cineol, tannins, triterpenes, flavonoids, resin, eugenol, malic acid, fat,
cellulose, chlorophyll, mineral salts, and a number of other fixed substances.[11]
MATERIAL AND METHODS
(i) Sample collection
The leaf samples were collected from the guava trees growing in fruit gardens at
Suddhowala, Dehra Dun, Uttarakhand. Random leaf samples were collected in plastic zip
lock bags.
(ii) Preparation of the extract
The collected leaves of P. guajava were air dried in shade at room temperature and milled to
a coarse powder. The obtained dried powder was subjected to continuous extraction with
80% ethanol and distilled water in a Soxhlet apparatus. The powdered plant material was
packed in a tumble made of filter paper. It was extracted with ethanol for 20 cycles. The
extract thus obtained was concentrated in a flask evaporator under reduced pressure and
controlled temperature. The obtained residue was green color, thick and sticky paste. The
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extract was stored in the refrigerator 4oC.
(iii) Phytochemical Screening of Psidium guajava leaf Extract
Phyto-chemical tests for the screening and identification of bioactive chemical constituents in
the guava leaves were carried out with the extracts using standard procedure as described
elsewhere.[12-14] For each test, 1ml of each solvent extract was used for analysis, in exception
for the saponin test in which 3ml solvent extract was used.
(a) Test for the presence of alkaloids

Wagner's test - Test solution with Wagner‟s reagent gives brown coloured precipitate in
presence of alkaloids.

Mayer's test - Test solution with Mayer's reagent gives cream coloured precipitate in
presence of alkaloids. Mayer's reagent is an alkaloidal precipitating reagent used for the
detection of alkaloids in natural products. Mayer‟s reagent is freshly prepared by dissolving a
mixture of mercuric chloride (1.36 g) and potassium iodide (5.00 g) in water (100.0 ml).
Most alkaloids are precipitated from neutral or slightly acidic solution by Mayer‟s reagent
(potassiomercuric iodide solution) to give a cream coloured precipitate.
(b) Test for the presence of carbohydrates and glycosides

Fehling's Test - Equal volume of Feling‟s solution A and B were added to make the test
solution. The mixture gives yellow red precipitate denoting the presence of carbohydrates.
(c) Test for the presence of saponins

Foam test - 1ml of extract was diluted with distilled water to 20 ml and shake in a
graduated cylinder for 15 minutes. A one cm layer of foam indicated the presence of
saponins.
(d) Test for the presence of proteins and amino acids

Million test – Test solution was treated with Million reagents gives white precipitate
which turns red upon gentle heating denoted the presence of proteins.

Ninhydrin test - Test solution treated with few drops of Ninhydrin reagent gives blue
coloured ring on the upper layer of the sample, if proteins are present.
(e)Test for steroids and tri-terpenoids

Salkowski test- Extract was treated with few drops of conc. sulfuric acid , shake well and
allowed to stand for some time, red colour appear at the lower layer indicated the presence of
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steroids and formation of golden yellow colored lower layer indicated the presence of triterpenoids.
(f) Test for tannins and phenolic compounds

Ferric chloride test- Extract was treated with ferric chloride solution, blue colour was
appeared if hydrolysable tannin was present and green colour was appeared if condensed
tannin was present.
(iv) Panel of Microorganisms
Bacterial strains were procured from Microbial Test Culture Collection (MTCC),
Chandigarh, India. The accession no. of Klebsiella, Proteus and Pseudomonas are MTCC7162, MTCC- 443 and MTCC-2488 respectively. Each bacterial suspension turbidity is then
compared to that of the 0.5 McFarland standard solution (containing about 1.5 × 108
CFU/mL).
(v) Antibacterial Activity
Antimicrobial susceptibility testing was done using the well-diffusion method according to
the standard of the National Committee for Clinical Laboratory Standards15. The plant
extracts were tested on Mueller Hinton II plates to detect the presence of antibacterial
activity. Prior to streaking the plates with bacteria, 5mm diameter wells were punched into
the medium using a sterile borer. All plates were inoculated with the test bacterium which has
been previously adjusted to the 0.5 McFarland standard solution; a sterile cotton swab was
dipped into the suspension, rotated several times, and pressed firmly on the inside wall of the
tube above the fluid level removing excess inoculum. The surface of the agar plate was
streaked over the entire sterile agar surface rotating the plate to ensure an even distribution of
inoculum with a final swab around the rim. The plates were allowed 3 to 5 min to dry the
excess moisture. 50µL aliquots of each test extract was dispensed into each well after the
inoculation of the plates with bacteria. The wells were also arranged in a triangle formation,
2 inches apart. The same extract was used on each plate, with a total of three plates used for
each extract for selecting bacterium. For each bacterial strain, controls were maintained
where pure solvents were used instead of the extract. The plates were sealed with paraffin
film, labeled, and incubated at set to 37°C. After 24 hours of incubation, each plate was
examined for inhibition zones. Inhibition zone was measured in mm.
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RESULTS
Phytochemical Analysis. Table 1 Summarizes the phyto-chemical screening of chemical
constituents of guava extracts on qualitative basis. The results revealed the presence of
several active compounds in the two different extracts. As the table shows, ethanol extracts
indicate the presence of Alkaloids, Protein & Amino acids, Saponins, Phenolic compounds,
Quinones and Flavonoids, but absence of Carbohydrates and Steroids. Aqueous extract
exhibited the presence of Alkaloids, Protein & Amino acid, Saponins, Phenolic compounds,
Steroids, and Quinones but absence of Carbohydrates and Flavonoids. The analysis of the
plant extract revealed the presence of phyto-chemicals which are known to exhibit medicinal
properties.
Table 1: Qualitative phyto-chemical screening of Psidium guajava extracts (+ Positive, Negative)
S. No.
1
2
3
4
5
6
7
8
Chemical
Constituents
Phytochemical test
Mayer‟s test
Wagner‟s test
Carbohydrates
Fehling test
Protein & Amino acid Ninhydrin test
Saponins
Foam test
Phenolic compounds
Ferric chloride test
and tannins
Steroids
Salkowski test
Quinones
Flavonoids
Alkaloids
Ethanol extract
Aqueous extract
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Antibacterial Activity: The results of the present study showed effective antibacterial
activity of aqueous and ethanol extracts of P. guajava by well diffusion method against
bacterial strains of Klebsiella, Proteus and Pseudomonas spp. The Klebsiella was inhibited by
both the extracts but aqueous extract of P. guajava showed greater inhibitory zone against this
bacterial strain at concentration of 250 mg/ml, i.e., 15.66±3.23 mm as compared to ethanol
extract, i.e., 12.5±3.06 mm (Table 2). Similar result was observed with Proteus, the zone of
inhibition is greater with aqueous extracts, i.e., 13.88±3.04 mm at concentration of 250
mg/ml as compared to ethanol extract that is 8.5±2.06 mm. The growth of Pseudomonas was
inhibited by the aqueous extract at concentration of 250 mg/ml and the zone of inhibition is
8.47±2.37 mm, the ethanol extract showed lesser zone of inhibition with same bacteria, i.e.,
4.01±1.63mm.
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Table 2: Antibacterial activity of aqueous and ethanol extracts of Psidium guajava
Plant extracts
Aqueous
Ethanol
Zone of inhibition (in mm)
Klebsiella sp. Pseudomonas sp. Proteus sp.
15.66±3.23
8.47±2.37
13.88±3.04
12.5±3.06
4.01±1.63
8.5±2.06
DISCUSSION
The results of phyto-chemical tests revealed the presence of bioactive compounds which are
known to exhibit antibacterial activities. The substances like tannins and polyphenolic
compounds are reported to bind to proline rich protein that interferes with protein synthesis
and exhibit antibacterial activity.[12, 16-18] Flavanoinds, another compound present in the guava
leaf exhibited antibacterial activity reported elsewhere.[19] The glycosides like saponins also
have been found to have inhibitory effects on many bacteria.[17] The phyto-chemical analysis
revealed the presence of such compounds, which are reported elsewhere.[17,19] as antibacterial
agents, may be responsible for the inhibitory actions on growth of bacteria of ethanol and
aqueous extracts of P. guajava.
In the present study, the results of the antibacterial activities of aqueous and ethanol extract
guava leaf showed that aqueous extract is more effective as compared to ethanol extract. It
has been reported elsewhere[20,21] that gram negative bacteria are usually resistant to plant
extract. However, antibacterial activities of guava leaf extracts against gram negative bacteria
also reported elsewhere.[22] The results of our present study showed both ethanol and aqueous
extracts of guava leaf are effective against gram negative bacteria. Climatic and geographical
variations and/or time of collection of leaf can be responsible for variations in exhibiting
inhibitory activities against test bacteria.
CONCLUSION
The results of the present study indicate better inhibitory action of aqueous extract of P.
guajava. It may be inferred from the results that those phyto-chemicals which are responsible
for inhibition of growth of bacteria under study extracted better in aqueous extraction as
compared to ethanol extraction.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Arun Kumar, Director and Dr. C.S. Pandey, Head, Department
of Agriculture, Dolphin (P.G.) Institute of Biomedical and Natural Sciences, Dehra Dun for
their valuable suggestions and advice during preparation of this manuscript. The authors also
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wish to acknowledge the management of this institute for providing all the necessary
facilities for the present study.
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