International Journal of Health Sciences and Research

www.ijhsr.org ISSN: 2249-9571

Original Research Article

Antifungal and Antibacterial Property of Guava

(Psidium guajava) Leaf Extract: Role of
Phytochemicals
Manika Das1, Subhagata Goswami2
1
Assistant Professor, 2Postgraduate Student
Department of Food Science and Nutrition Management, J.D Birla Institute, 11 Lower Rawdon Street,
Kolkata- 700020, India.
Corresponding Author: Manika Das

ABSTRACT

The traditional medical practice is an integral part of the culture and the interpretation of health by
indigenous populations in most of the world. Guava (Psidium guajava L.) leaves have traditionally been
used to manage several diseases such as rheumatism, diarrhea, diabetes mellitus, and cough. In this present
investigation antifungal and antibacterial property of guava leaves were estimated using Bacillus subtilis
(Gram positive bacterial strain), Escherichia coli (Gram negative bacterial strain), Saccharomyces
cerevisiae (Yeast, fungal strain) and Aspergillus niger (Mould, fungal strain) strains. The growth of gram
positive bacteria and fungal strains were inhibited strongly, whereas gram negative bacterial strain
displayed less sensitivity against the antimicrobial (antifungal and antibacterial) property of guava leaf
extract. Zone inhibition assay also confirmed the result. Phytochemical analysis (qualitative and
quantitative) revealed that guava leaf extract was rich in wide range of poly phenols. It was found that
guava leaves are rich in phenols, flavonoids and tannins whereas components like alkaloids, flavonoids,
saponins and triterpenes are present in comparatively lesser amounts. As polyphenols have strong
antimicrobial property, it can be concluded that rich source of phenols, flavonoids and tannins are the
probable cause of anti microbial property of guava leaves.
Keywords: Antifungal, antibacterial, guava leaf, phytochemical, polyphenol

1. INTRODUCTION Psidium guajava L., popularly

Study of traditional medical practice, known as guava, is a small tree belonging to
is an integral part of the culture and the the myrtle family (Myrtaceae). [4] Native to
interpretation of health by indigenous tropical areas from southern Mexico to
populations in many parts of the world. [1] northern South America, guava trees have
For example, Indian Ayurveda and been grown by many other countries having
traditional Chinese medicine are among the tropical and subtropical climates, thus
most enduring folk medicines still practiced. allowing production around the world. [5]
These systems try to promote health and Traditionally, preparations of the leaves
improve the quality of life, with therapies have been used in folk medicine in several
based on the use of indigenous drugs of countries, mainly as anti-diarrheal remedy.
natural origin. [2] Given that plants have [6]
Moreover, other several uses have been
been widely used as herbal medicines, described elsewhere on all continents, with
several approaches are now being carried the exception of Europe. [6-8] Depending
out to discover new bioactive compounds. upon the illness, the application of the
[3]
remedy is either oral or topical. The

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Manika Das et.al. Antifungal and Antibacterial Property of Guava (Psidium Guajava) Leaf Extract: Role of
Phytochemicals

consumption of decoction, infusion, and gm) and agar (10gm) were dissolved in
boiled preparations is the most common distilled water (500 ml), in a conical flask.
way to overcome several disorders, such as The mixture was sterilised in an autoclave.
rheumatism, diarrhea, diabetes mellitus, and 2.4. Estimation of antimicrobial activity
cough in India, China, Pakistan, and of guava leaf extract
Bangladesh, [6-9] while in Southeast Asia the The antimicrobial activity of the
decoction is used as gargle for mouth ulcers. methanol extract of guava leaves, was
[6,8,9]
For skin and wound applications, estimated against four microbial strains:
poultice is externally used in Mexico, Bacillus subtilis (Gram positive bacterial
Brazil, Philippines, and Nigeria. [6-9] In strain) [MTCC No: 441], Escherichia coli
addition, chewing stick is used for oral care [MTCC No: 1696](Gram negative bacterial
in Nigeria. [9] strain), Saccharomyces cerevisiae [MTCC
Currently, there is increasing interest No: 3090](Yeast, fungal strain) and
in studying of plants regarding their Aspergillus niger [MTCC No: 10180]
chemical components of bioactive (Mould, fungal strain).
compounds, their effects on several 2.4.1. Examination of microbial (fungus
diseases, and their use for human health as and bacteria) growth in the presence and
functional foods and/or nutraceuticals. [10] In absence of Guava leaf extract
recent years, guava leaves tea and some For each microbial strain, four petri
complementary guava products are available plates were used, of which one was used as
in several shops in Japan as well as on the ‘Control’ plate, while the rest were used as
Internet, [11] because guava leaf phenolic ‘Test’ plates containing different amount of
compounds have been claimed to be food Guava leaf extract as for example 0.5 ml,
for specified health use (FOSHU), since 1ml and 2ml, respectively along with
they have beneficial health effects related to nutrient agar. The petri plates were streaked
the modulation of blood–sugar level. [12] In with the above-mentioned microbial strains
this present investigation we tried to find and were incubated at 37ºC. Microbial
out the antifungal and antibacterial property growths were observed after 24 hours.
of guava leave and the probable cause 2.4.2. Estimation of zone inhibition using
behind it. Well Diffusion method
Before preparing the petri plates,
2. METHODOLOGY liquid cultures of the microbial strains were
2.1. Preparation of guava leaf powder: prepared by inoculating lactose broth with
Fresh guava leaves were collected previously prepared sub cultures and
and air dried for 10 days. The dried leaves incubating it for 24 hours at 37C to observe
were then crushed and churned in a blender the turbidity. [13] The nutrient agar
to form a coarse powder. The powder was containing petri plates were inoculated with
collected in an air tight container and stored these liquid cultures using spread plating
in a cool, dry place, away from sunlight. method.
2.2. Preparation of methanol extract of For each microbial strain, one petri
guava leaf powder: plate was kept aside as ‘Control’ plate while
The methanol extract was prepared another was used as ‘Test’ plate which
by mixing 20 grams of guava leaf powder contained Guava leaf extract in three wells,
with 100 ml of methanol, which was kept at concentrations of 50 µl, 100 µl and 150
for 3 days in a cool, dark place along with µl, respectively. The petri plates were
occasional stirring. After 3 days, the extract incubated at 37ºC and zone of inhibition
was filtered into sterilised test tubes. was observed after 24 hours.
2.3. Preparation of nutrient agar (500ml): 2.5. Qualitative analysis of the
Weighed amounts of peptone (2.5 phytochemical content of guava leaf
gm), glucose (5 gm), and beef extract (0.75 extract

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Manika Das et.al. Antifungal and Antibacterial Property of Guava (Psidium Guajava) Leaf Extract: Role of
Phytochemicals

Chemical tests for the screening and 2.6. Quantitative analysis of phenol
identification of bioactive chemical content of guava leaf extract:
constituents in the guava were carried out The total phenolic content of guava
with the extracts using the standard leaf extract was determined using a
procedure as described. [14] spectrophotometer and Folin Ciocalteu’s
2.5.1. Test for alkaloids: The leaf extracts reagent. The Folin reagent was first
of guava were dissolved and filtered in prepared at a dilution of 10% using distilled
dilute hydrochloric acid. Wagner’s reagent water, and to it, 5 ml of the guava leaf
was prepared by mixing 6 gm of potassium extract was mixed. Thereafter, anhydrous
iodide and 2 gm of iodine in 100 ml of sodium carbonate was prepared at a
distilled water, and then added to the concentration of 75%, and 4ml of it was
previously obtained filtrate. The presence of added to the Folin mixture, hence producing
alkaloids would be confirmed with the a blue coloured solution. A blank was
appearance of reddish-brown precipitate. prepared and the resulting mixtures were
2.5.2. Test for anthraquinones: 0.2 ml of shaken vigorously and incubated at 40C for
guava leaf extracts were taken, to which 2 30 minutes. The resulting optical densities
ml of chloroform was added. The mixture were measured at 765 nm using a
was shaken and filtered, and was mixed spectrophotometer. The absorbance against
with 10% ammonia solution. The presence mg of gallic acid was measured and the
of anthraquinones would be confirmed with graph obtained was used to determine the
the presence of bright pink precipitate. phenol content of guava leaf extract. [14]
2.5.3. Test for flavonoids: 0.5 ml of guava 2.7. Quantitative analysis of tannin
leaf extracts were taken, to which few drops content of guava leaf extract
of 10% sodium hydroxide was added. The Tannin content of guava leaf extract
appearance of bright yellow colour, which was determined spectrophotometrically
will disappear with the addition of dilute using Folin Ciocalteu’s reagent. Standard
acid, would confirm the presence of Tannic acid solution was prepared by
flavonoids. mixing 100 mg Tannic acid in 1000 ml
2.5.4. Test for phenols: 0.5 ml of guava water. This solution was pipetted in 100ml
leaf extracts were taken, to which few drops flasks containing 75 ml distilled water in
of 10% ferric chloride solution was added. amounts of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
The presence of phenol would be confirmed 0.8, 0.9 and 1.0 ml. To each mixture, 5 ml
with the appearance of bluish black colour. of Folin reagent was added, along with 10ml
2.5.5. Test for saponins: The guava leaf was sodium carbonate solution (prepared by
extract was taken in a test tube and shaken dissolving 35 gm anhydrous sodium
vigorously. Formation of a stable foam carbonate in 100ml water). The mixtures
would confirm the presence of saponins. was shaken well and incubated for 30
2.5.6. Test for tannins: 0.5 ml of guava leaf minutes at 40C. The absorbance against mg
extracts were taken, to which 10% sodium of tannic acid was plotted and the graph
chloride solution containing 1% gelatin obtained was used to determine the
solution was added. The presence of tannins concentration of tannin in guava leaf
would be confirmed with the appearance of extract. [15]
white precipitate. The sample was prepared by
2.5.7. Test for triterpenes: to guava leaf pipetting 1 ml of guava leaf extract in a
extracts, chloroform was added and filtered. flask containing 80 ml water. To it, 5 ml of
To it, few drops of concentrated sulphuric Folin reagent was added along with 75%
acid was added, shaken and kept aside. The sodium carbonate solution. The volume was
presence of triterpenes was would be made up and the mixture was incubated at
confirmed with the appearance of golden 40C for 30 minutes against blank and the
yellow colour. absorbance was measured.

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Manika Das et.al. Antifungal and Antibacterial Property of Guava (Psidium Guajava) Leaf Extract: Role of
Phytochemicals

2.7. Quantitative analysis of flavonoid 3.1. Estimation of antimicrobial activity

content of guava leaf extract (antifungal and anti bacterial) of guava
The flavonoid content was leaf extract
determined according to the method The antimicrobial activity of methanol
described by Ordonez et al. [16] Briefly, 0.5 extract of guava leaves was assessed against
mL of the extract was mixed with 0.5 mL of four microbial strains: Bacillus subtilis
2% aluminum chloride (prepared in (Gram positive bacterial strain), Escherichia
ethanol).The mixture was incubated for 1 h coli (Gram negative bacterial strain),
at room temperature, after which the Saccharomyces cerevisiae (Yeast; fungal
absorbance was read at 420 nm. strain) and Aspergillus niger (Mould; fungal
Development of yellow color indicated the strain).
presence of flavonoid. Total flavonoid 3.1.1. Examination of microbial (fungus
content was calculated as mg/g of quercetin and bacteria) growth in the presence and
equivalent using the calibration curve. absence of Guava leaf extract
The results of all microbial and chemical After 24 hours of incubation, the microbial
analysis were expressed as mean ± standard growth was observed for bacterial as well
deviation of triplicate analyses. the fungal strains (Table: 1)
3. RESULTS & DISCUSSION

Table 1: Estimation of antimicrobial activity of guava leaf extract using pour plating technique
Microbial growth observed
Microbial Strain Amount of Guava leaf extract
Control 0.5 ml 1 ml 2 ml
Bacillus subtilis +++ + - -
Escherichia coli +++ ++ ++ -
Saccharomyces cerevisiae +++ + - -
Aspergillus niger +++ + - -

++ : High Growth, +: Low growth, - : No growth

The petri plates streaked with gram observed in the petri plate containing 2 ml
positive strain and fungal strain plenty of guava leaf extract. The result of this study
growth was observed in the ‘Control’ plate. indicated that, gram negative bacterial strain
In the petri plate containing 0.5 ml of guava Escherichia coli, was less sensitive towards
leaf extract, less microbial growth was the antimicrobial activity of guava leaf
observed. No microbial growth was extract.
observed in the petri plates containing 1 ml 3.1.2. Estimation of zone inhibition by
and 2 ml of guava leaf extract, respectively. Well Diffusion method
In the petri plates streaked with gram After 24 hours of incubation, the Zones of
negative bacterial strains (Escherichia coli), Inhibition were measured for the bacterial
abundant growth was observed in the and fungal strains (Table: 2). Results
‘Control’ petri plate, as well in the petri showed that zone of inhibition were much
plates containing 0.5 ml and 1 ml guava leaf less in case of gram –ve bacteria compared
extract, respectively. Negligible growth was to gm +ve bacteria and fungal strain.

Table 2: Zone of inhibition of bacterial and fungal strains against different concentration of guava leaf extract
Microbial strain Zone of Inhibition (cm)
50 µl Guava leaf extract 100 µl Guava leaf extract 150 µl Guava leaf extract
(mm) (mm) (mm)
Bacillus subtilis 14 ± 3.2 16 ± 4.3 20 ± 4.2
Escherichia coli 08 ± 2.6 10 ± 2.5 13 ± 3.6
Saccharomyces cerevisiae 16 ± 4.3 18 ± 3.3 24 ± 5.2
Aspergillus niger 15 ± 4.2 19 ± 4.4 21 ± 5.4
Inhibition zones are the mean including borer (5 mm) diameter ± standard deviation

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Manika Das et.al. Antifungal and Antibacterial Property of Guava (Psidium Guajava) Leaf Extract: Role of
Phytochemicals

3.2. Qualitative analysis of the outer membrane, which consists of

phytochemical content of guava leaf lipopolysaccharide, a peptidoglycan cell
extract wall consisting of partially cross linked
Phytochemical analysis of guava leaf extract peptide chains, and an inner or cytoplasmic
showed that phenols, flavonoids and tannins membrane. [17]
are present in significantly large amounts Gram positive bacteria are generally
whereas components like alkaloids, devoid of this outer membrane as compared
saponins and triterpenes are present in to gram negative bacteria. The concerned
comparatively lesser amounts in guava leaf outer membrane acts as a barrier with
extract (Table: 3). limited permeability, which prevents the
Table 3: Qualitative Analysis of Guava Leaf extract
penetration of certain antibiotic components
Qualitative Analysis of Guava Leaf extract and drugs into the cell. The presence of this
Compound tested Result outer membrane is one of the major reasons
Alkaloids +
Anthroquinones - contributing towards the increased antibiotic
Flavonoids ++ resistance of Gram negative bacteria, as
Phenols ++
Saponins +
compared to their gram positive
Tannins ++ counterparts. [18]
Triterpenes + The value of edible/ medicinal plants
++: Strongly Detected, +: Detected; −: Not Detected
to mankind is well established, as numerous
3.3. Quantitative analysis of the discoveries have shown that plants' extracts
phytochemical content of guava leaf contain not only minerals and primary
extract metabolites, but also a wide range of
Since qualitative analysis of guava leaf secondary metabolites with great therapeutic
showed that it is very rich in phenol, tannin efficiencies.
and flavonoid, quantitative analysis Phytochemical analysis of guava
(spectrophotometrically) were performed leave showed the guava leaves are rich in
for phenol, tannin and flavonoid. (Table: 4) flavonoids, phenols and tannins whereas
components like alkaloids, saponins and
Table 4: Quantitative analysis phenol, tannin and flavanoid. triterpenes are present in comparatively
of Guava leaf extract lesser amounts in guava leaf extract. It is
Compound analysed Concentration (mg/ml)
Tannin 3.07 ± 0.6 well known that along with powerful anti-
Phenol 2.62 ± 0.7 inflammatory, free radical scavenging,
Flavanoids 0.9 ± 0.3
antioxidant and anti-carcinogenic properties,
polyphenolic compounds contain potent
4. DISCUSSION antimicrobial activity. [19] Inhibition of
Overall result indicated that Gram
RNA, DNA, and depolarization of
negative bacteria (Escherichia coli)
cytoplasmic membrane and inhibition of
exhibited less sensitivity compared to the
macromolecular synthesis are some of the
Gram positive bacteria (Bacillus subtilis)
underlying antimicrobial mechanisms of
towards the antimicrobial property of Guava
action of polyphenolic compounds. [20]
leaves, whereas the fungal strains showed
Evidences also suggested that phenols show
strong sensitivity towards the antimicrobial
anti-fungal activity by inhibiting ergosterol
property of guava leaves. Gram negative
which is the component of fungal cell
bacteria are less sensitive to antimicrobial
membrane and glucosamine, a growth
components as compared to Gram positive
indicator present only in the fungal cells of
bacteria, may be due to a difference in their
some genera and some other proteins. [21]
respective cell wall structures.
Tannins are water-soluble chemical
The cell wall of Gram negative
compounds found widely in the plant
bacteria consist of three distinct layers,
kingdom, which exhibit strong antimicrobial
which act as an envelope towards certain
property. The various mechanism which are
bioactive components. These include: an
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Manika Das et.al. Antifungal and Antibacterial Property of Guava (Psidium Guajava) Leaf Extract: Role of
Phytochemicals

present in tannins to inhibit microbial 3. Fabricant DS, Farnsworth NR. The value of
growth include deprivation of iron via iron plants used in traditional medicine for drug
chelation, disturbance in the metabolic discovery. Environ. Health Perspect. 2001;
activities of microbes via inhibition of 109: 69–75.
4. Morton JF. Fruits of Warm Climates;
oxidative phosphorylation, deprivation of
Creative Resource Systems, Inc.:
essential compounds responsible microbial Winterville, NC, USA, 1987.
growth and also, inhibition of enzymes 5. Salazar DM, Melgarejo P, Martínez R,
required in the extracellular cytoplasmic Martínez, JJ, Hernández F, Burguera M.
membrane. [22] Tannin act as a antimicrobial Phenological stages of the guava tree
agent by the mechanism such as: (Psidium guajava L.). Sci. Hortic. 2006;
extracellular enzyme inhibition, deprivation 108: 157–161.
of substratum, inhibition of oxidative 6. Gutiérrez RMP, Mitchell S, Solis RV.
phosphorylation. [23] From the above Psidium guajava: A review of its traditional
discussion it can be concluded that rich uses, phytochemistry and pharmacology. J.
source of wide range of poly phenolic Ethnopharmacol. 2008; 117: 1–27.
7. Shruthi SD, Roshan A, Sharma S, Sunita S.
compounds (Phenol, flavonoids and tannin)
A review on the medicinal plant Psidium
may be the most probable cause of the
guajava Linn. (Myrtaceae). J. Drug Deliv.
antimicrobial property of guava leaves. Ther. 2013; 3: 162–168.
8. Morais-Braga MFB, Carneiro JNP,
CONCLUSION Machado AJT, Dos Santos ATL, Sales DL,
From this investigation it can be Lima LF, Figueredo FG, Coutinho HDM.
concluded that Guava leaves posses strong Psidium guajava L., from ethnobiology to
antimicrobial (antibacterial and antifungal) scientific evaluation: Elucidating bioactivity
property although the gram negative against pathogenic microorganisms. J.
bacteria showed less sensitivity towards the Ethnopharmacol. 2016; 194: 1140–1152.
antimicrobial property of guava leaves. 9. Sanda KA, Grema HA, Geidam YA, Bukar-
Kolo YM. Pharmacological aspects of P.
Phytochemical analysis showed that Guava
guajava: An update. Int. J. Pharmacol. 2011;
leaves are rich in wide range of 7: 316–324.
polyphenolic compounds (Phenol, 10. Bernal J, Mendiola, JA, Ibáñez E, Cifuentes
flavonoids and tannin). As the polyphenolic A. Advanced analysis of nutraceuticals. J.
compounds posses the antimicrobial Pharm. Biomed. Anal. 2011; 55: 758–774.
property, it may be the most probable cause 11. Matsuda K, Nishimura Y, Kurata N, Iwase
of antifungal and antibacterial property of M, Yasuhara H. Effects of continuous
guava leave. Not only that for the ingestion of herbal teas on intestinal CYP3A
antimicrobial property, guava leaves can be in the rat. J. Pharmacol. Sci. 2007; 103:
used in pharmaceutical industry as well as 214–221.
in food industry as bio preservative. For 12. Arai S, Yasuoka A, Abe. K. Functional food
science and food for specified health use
their detail mechanism of action further
policy in Japan: State of the art. Curr. Opin.
investigations are required.
Lipidol. 2008; 19: 69–73.
13. Biswas B, Rogers K, McLaughlin F,
REFERENCES
Daniels D, Yadav A. Antimicrobial
1. Anyinam C. Ecology and ethnomedicine:
Activities of Leaf Extracts on Two Gram-
Exploring links between current
Negative and Gram-Positive Bacteria.
environmental crisis and indigenous medical
International Journal of Microbiology.
practices. Soc. Sci. Med. 1995; 40: 321–
2013; 5 (6): 1 – 7.
329.
14. Sagbo IJ, Afolayan AJ, Bradley G.
2. Patwardhan B, Warude D, Pushpangadan P,
Antioxidant, antibacterial and
Bhatt N. Ayurveda and traditional Chinese
phytochemical properties of two medicinal
medicine: A comparative overview. Evid.
plants against the wound infecting bacteria.
Based Complement. Altern. Med. 2005; 2:
Asian Pacific Journal of Tropical
465–473.
Biomedicine. 2017; 7 (9): 817 – 825.

International Journal of Health Sciences & Research (www.ijhsr.org) 44

Vol.9; Issue: 2; February 2019
Manika Das et.al. Antifungal and Antibacterial Property of Guava (Psidium Guajava) Leaf Extract: Role of
Phytochemicals

15. Doss A, Dhanabalan R. Antibacterial 20. Ozcelik B, Orhan DD, Ozgen S, Ergun, F.
activity of tannins from the leaves of Antimicrobial Activity of Flavonoids
Solanum trilobatum Linn. Indian Journal of against Extended-Spectrum β-Lactamase
Science and Technology. 2009: 2 (2): 41 – (ESβL)-Producing Klebsiella pneumoniae.
44. Tropical Journal of Pharmaceutical
16. Ordonez AA, Gomez JD, Vattuone MA, Isla Research. 2008; 7 (4): 1151 – 1157.
MI. Antioxidant activities of Sechium edule 21. Coppo E, Marchese A. Antibacterial
(jacq). swart extracts. Food Chem 2006; 97: Activity of Polyphenols. Current
452-8. Pharmaceutical Biotechnolog.2014; 15 (4):
17. Exner M, Bhattacharya S, Christiansen B, 380 – 392.
Gebel J, Trautmann M.. Antibiotic 22. Mailoa NM, Mahendradatta M, Laga A,
resistance: What is so special about Djide N. Tannin Extract of Guava Leaves
multidrug-resistant Gram-negative bacteria? (Psidium guajava L.) Variation with
GMS Hygiene and Infection Control. 2017; Concentration Organic Solvents.
12 (5): 1 – 24. International Journal of Scientific and
18. Miller SI. Antibiotic Resistance and Technology Research. 2013; 2 (9): 106 –
Regulation of the Gram-Negative Bacterial 110.
Outer Membrane Barrier by Host Innate 23. Ansari MA, Amiya A, Fatima Z, Hameed,
Immune Molecules. American Society for S. Natural Phenolic Compounds: A
Microbiolog.2016: 7 (5): 1 – 3. Potential Antifungal Agent. Microbial
19. Xie Y, Yang W, Tang F, Chen X., Ren L. Pathogens and Strategies for combating
Antibacterial Activities of Flavonoids: them: Science, Technology and Education.
Structure-Activity Relationship and 2013; 7 (8): 1189 – 1195.
Mechanism. Current Medical Chemistry.
2015; 22 (7): 132 – 149.

How to cite this article: Das M, Goswami S. Antifungal and antibacterial property of guava
(Psidium guajava) leaf extract: role of phytochemicals. Int J Health Sci Res. 2019; 9(2):39-45.

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