Inte r na t io na l J o ur na l o f A pp lie d Re se a r c h 2 0 2 0 ; 6 (5 ): 1 0 6 -1 1 0

ISSN Print: 2394-7500

ISSN Online: 2394-5869
Impact Factor: 5.2
Phytochemical and antimicrobial activities of leaf
IJAR 2020; 6(5): 106-110
www.allresearchjournal.com
extract of Guava (Psidium guajava L.)
Received: 26-03-2020
Accepted: 28-04-2020
Pooja Ratnakaran, Anuja Anirudha Barve, Kavita Avinash Patnekar,
Pooja Ratnakaran Nayan Chandulal Patil, Neha Madhukar Udmale, Sapna Ramchandran
Department of Biotechnology,
BK Birla College Autonomous,
and Annika Durve-Gupta
Kalyan, Dist. Thane,
Maharashtra, India Abstract
Medicines and products that are produced from natural sources have been gaining a lot of attention.
Anuja Anirudha Barve Various fruits and their extracts have been found to exhibit antimicrobial activity. Guava (Psidium
Department of Biotechnology, guajava L.) is believed to have active components that help in treating various diseases and has been
BK Birla College Autonomous, used in folk medicine. In this study, the phytochemical analysis of the aqueous and methanolic extracts
Kalyan, Dist. Thane,
of guava leaves was carried out which showed the presence of flavonoids, saponins, phenols,
Maharashtra, India
glycosides and terpenoids. Minimum Inhibitory Concentration (MIC) of the leaf extracts was checked
Kavita Avinash Patnekar towards Gram-positive and Gram-negative bacterial isolates and was found to lie in the range of 30 to
Department of Biotechnology, 60 mg/ml. The leaf extracts were used for determining their antimicrobial activity on S. aureus and E.
BK Birla College Autonomous, coli using agar well diffusion assay method. Future experiments have to be conducted to evaluate the
Kalyan, Dist. Thane, effects of guava leaves on other bacterial isolates.
Maharashtra, India
Keywords: Guava leaves, Psidium guajava L., antimicrobial activity, phytochemical analysis, MIC.
Nayan Chandulal Patil
Department of Biotechnology,
1. Introduction
BK Birla College Autonomous,
Kalyan, Dist. Thane, For the treatment of various diseases like pneumonia, diarrhoea, ulcers, bronchitis, colds and
Maharashtra, India diseases of the respiratory tract, the World Health Organization (WHO) has catalogued more
than 20,000 plant species with medicinal properties (Gonçalves et al., 2008) [6]. This suggests
Neha Madhukar Udmale that plants can be used to inhibit the growth of pathogens since they manifest compounds
Department of Biotechnology, with relatively high levels of antimicrobial action (Kim and Fung, 2004) [9]. The study of
BK Birla College Autonomous,
Kalyan, Dist. Thane, traditional medicines is an integral part of the culture in many parts of the world like for
Maharashtra, India example Indian Ayurveda is among the many other enduring folk medicines which are still
practiced. These systems try to improve the quality of life, promote health with therapies
Sapna Ramchandran based on the use of natural materials. Several approaches are now being carried out to
Department of Biotechnology, discover new bioactive compounds as plants have been widely used as herbal medicines (Das
BK Birla College Autonomous,
Kalyan, Dist. Thane, and Goswami, 2019) [5].
Maharashtra, India Guava (Psidium guajava L.) is an evergreen tree with leaves up to 2-6 inches in length and 1-
2 inches in width (Fig. 1). The leaves of the tree are dull green in colour, when crushed give
Annika Durve-Gupta aroma and appear stiff but coriaceous with pronounced veins. The guava leaves help in
Department of Biotechnology, fighting against pathogens, maintaining blood glucose level and even aiding in weight loss
BK Birla College Autonomous,
Kalyan, Dist. Thane,
due to the bioactive components present in them (Biswas et al., 2013) [3].
Maharashtra, India

Correspondence Author:
Annika Durve-Gupta
Department of Biotechnology,
BK Birla College Autonomous,
Kalyan, Dist. Thane,
Maharashtra, India Fig 1: Guava fruit
~ 106 ~
International Journal of Applied Research http://www.allresearchjournal.com

Scientific classification: (Maurya et al., 2018) [11] extract was filtered and then subjected to evaporation by
Kingdom: Plantae keeping the flask in boiling water bath to evaporate the
Division: Magnoliophyta entire methanol. The dried residue was scraped and diluted
Class: Magnoliopsida with distilled water. Both the extracts were stored at 4°C
Order: Myrtales until further use (Biswas et al., 2013) [3].
Family: Myrtaceae
Genus: Psidium L. Qualitative analysis of the phytochemical content in the
Species: Psidium guajava L. leaf extract
Binomial name: Psidium guajava L. Phytochemical tests were done for the screening of bioactive
Many parts of the guava tree have been used in traditional compounds in the guava leaves with the help of the extracts
medicine to treat conditions like gastroenteritis, malaria, viz. Flavonoids (Lead Acetate Test), Saponins (Foam Test),
vomiting, diarrhoea, dysentery, ulcers, toothache, cough, Tannins (Ferric Chloride Test), Glycosides, Terpenoids
sore throat, wounds, inflamed gums, etc (Abdelrahim et al., (Salkowski Test), Phenols (Ferric Chloride Test), were
2002; Lutterodt et al., 1992) [1, 10]. The leaves of guava carried out to check the phytochemical constituents present
contain an essential oil rich in cineol, tannins, resin, in the leaf extracts (Biswas et al., 2013) [3].
uquenol, malic acid, fat, terpenoids, cellulose, chlorophyll,
mineral salts and several other fixed substances (Biswas et Test organisms
al., 2013) [3]. One of the many methods in which plants are The MIC and antimicrobial activity of the leaf extracts were
used in traditional remedies is to extract and consume tested against Escherichia coli and Staphylococcus aureus.
essential plant oils (Gonçalves et al., 2008) [6]. These bacterial cultures were procured from the Department
The efficient use of different parts of guava like leaves, of Biotechnology, B. K. Birla College, Kalyan. For regular
seeds and peels may reduce the risk of their disposal as use, the organisms were cultured and maintained on sterile
pollutants since they are treated as wastes by the food Nutrient agar slants and stored in a refrigerator (Harley and
processing industries and eventually discarded. Since all the Prescott, 2002) [7].
parts of the plant have been used for different purposes like
hepatoprotection, antioxidant, antimicrobial, anti- Minimum inhibitory concentration
inflammatory, anti-spasmodic, anti-cancer, anti- The minimum concentration of any substance that inhibits
hyperglycemic, analgesic, anti-stomachache, anti-diarrhoea, the growth of the organism is known as the Mininmum
etc and shown to have positive effects on health it should be Inhibitory Concentration (MIC). The MIC of the extracts
researched more extensively (Barbalho et al., 2012) [2]. The was determined for the bacterial isolates viz. E. coli and S.
present study was carried out with an aim to evaluate the aureus. The concentration range of the leaf extracts
effectiveness of the Psidium guajava leaf extracts made (Methanol and Distilled water) were made in the range of 10
using aqueous solvent (distilled water) and organic solvent mg/ml to 100 mg/ml using Sterile plain Nutrient broth as the
(methanol) in inhibiting the growth of Escherichia coli and diluent. 0.1 ml of 24 hours old culture suspension (E. coli
Staphylococcus aureus. and S. aureus) was inoculated in the tubes respectively. A
positive control (Sterile plain nutrient broth inoculated with
2. Materials and methods test organism) and negative control (Sterile plain nutrient
The media components like Nutrient agar and Nutrient broth broth) were also kept. All the tubes were incubated at 37°C
used for this study were procured from Himedia, India. The for 24 hours (Puntawong et al., 2012) [15]. The lowest
chemicals used were of analytical grade. All the chemicals concentration that completely inhibited bacterial growth
and media were made in distilled water. after 24 hours of incubation was determined as the MIC
value of the extract (Nayak et al., 2019) [12].
Preparation of guava leaf powder
The leaf samples were collected from guava trees growing Antimicrobial susceptibility test
in different towns like Kalyan and Ambarnath. Random Antimicrobial susceptibility testing was performed using the
fresh leaf samples were collected into plastic bags. The well-diffusion/ agar cup method. In this method, the growth
collected leaf samples were thoroughly washed and sun- response is measured by the zone of inhibition of the test
dried for 24 hours. It was then grounded into a fine powder culture in response to different concentrations of the extract.
using a mixer grinder. The powder was kept in a clean The Sterile molten nutrient agar was seeded with 1ml of the
container and stored in a cool, dry place until further use 24 hours old test culture suspensions and the plates are
(Biswas et al., 2013) [3]. prepared. Wells were made in the solidified media using a
cork borer having an internal diameter of 6mm. The
Preparation of methanolic and aqueous (distilled water) methanolic extract (50 mg/ml for S. aureus and 30 mg/ml
extract for E. coli) and distilled water extract (30 mg/ml for S.
Two different leaf extracts were made, one in distilled water aureus and 20 mg/ml for E. coli) were added to the wells.
using hot water extraction method and the other in These values wewre based on the MIC results obatined.
methanol. The methanolic and aqueous extracts were Gentamicin (50 mg/ml) was used as a control. The plates
prepared in sterile 150 ml conical flask by mixing 10 grams were kept in a refrigerator for 10 minutes for pre-diffusion
of the leaf powder in 100 ml of methanol and boiling after which they were incubated at 370C for 24 hours. The
distilled water, respectively, making the final concentration added extract diffuses away from the well in a decreasing
of the extracts to 100 mg/ml. The flasks were then kept in a concentration gradient so that the susceptibility by the
shaker at 250C for 7 hours at 100 rpm. The aqueous extract seeded organism can be seen. The zone of inhibitions was
was filtered through a muslin cloth and transferred into measured after the incubation period (Khan et al., 2019).
sterile test tubes. In the case of the methanolic extract, the
~ 107 ~
International Journal of Applied Research http://www.allresearchjournal.com

3. Results and discussion Qualitative analysis of the phytochemical content in the

Preparation of extracts leaf extract
Guava leaves collected were powdered and suspended in The phytochemical analysis was carried out on a qualitative
solvents viz. methanol and distilled water (Fig. 2) and basis to determine the presence of chemical constituents in
subjected to shaker conditions at 250C for 7 hours at guava leaf extracts. The presence of bioactive compounds
100rpm. The solutions obtained after the incubation period was observed in both methanol and distilled water extracts.
were filtered and the filtrates obtained were used as an The methanol extract showed the presence of phenols,
extract. Effect of plant material depends on its origin, flavonoids, terpenoids, glycosides and saponins whereas
extraction technique, time, the temperature of extraction, distilled water extract showed the presence of all the
solvent concentration, variations, and polarity, quantity and phytochemicals except glycosides as shown in Fig. 3, Table.
secondary metabolite composition of an extract (Ncube et 1. These matched with the results of the phytochemical
al., 2006) [13]. analysis of guava leaf extract carried out by Das and
Goswami (2019) [5]. They showed that phenols, flavonoids
and tannins are present in large amounts whereas
components like triterpenes, alkaloids and saponins are
present in comparatively lesser amounts in guava leaf
extract (Das and Goswami., 2019) [5]. Flavonoids are active
compounds showing antibacterial properties. Terpenoids
have also been found to be potential antibacterial agents
against inhibiting bacteria inspite being mostly used for their
aromatic properties (Biswas et al., 2013) [3]. Phenols inhibit
ergosterol which is a component of fungal cell membrane
and glucosamine, a growth indicator present only in the
fungal cells, indicating the presence of anti-fungal activity.
Tannins which are water-soluble compounds, act as an
antimicrobial agent with the help of different mechanisms
like deprivation of substratum, inhibition of oxidative
phosphorylation, extracellular enzyme inhibition, etc. Hence
it can be concluded that the most probable cause of the
Fig 2: Methanolic (left) and distilled water (right) extract antimicrobial activity of guava leaves extract is the wide
range of polyphenolic compounds (Das and Goswami.,
2019) [5].

Fig 3: Phytochemical testings of plant extract

Table 1: Phytochemical analysis of Psidium guajava extracts

S. No. Extracts Saponins Flavonoids Terpenoids Glycosides Phenols
1. Methanol + + + + +
2. Distilled water + + + - +
Key: +: presence of the constituent, -: absence of the constituent

Minimum Inhibition Concentration different extracts (Aqueous and Methanolic) were

Minimum Inhibition Concentration is the lowest investigated to determine their Minimum inhibition
concentration of a compound at which the test micro- Concentrations against test micro-organisms viz. Escherichia
organism does not demonstrate any visible growth. Two coli and Staphylococcus aureus. The methanolic extract
~ 108 ~
International Journal of Applied Research http://www.allresearchjournal.com

showed MIC in the range of 30-40 mg/ml and 50-60 mg/ml presence of different active compounds present in them.
against E. coli and S. aureus respectively. While distilled Tannins might be the active compound in guava leaves that
water extract showed the result in the range of 20-30 mg/ml can be used to control bacterial resistance to antibiotics. The
and 30-40 mg/ml against E. coli and S. aureus respectively different factors like cropping areas of the herbs, the season,
as shown in Table. 2. Numerous studies have demonstrated extract, and extraction methods may affect the MIC value
that the guava leaf extract could inhibit the growth of E. coli (Chukiatsiri et al., 2019) [4].
with different MIC values. This can be attributed to the

Table 2: Minimum Inhibition Concentration

S. No. Test organisms Methanol (mg/ml) Distilled water (mg/ml)

1. Escherichia coli 30-40 20-30
2. Staphylococcus aureus 50-60 30-40

Antimicrobial Susceptibility Test molecules (active components) of the solvent extracts may
The antimicrobial activity of the Guava leaves was studied affect the inhibition zone values (Power, 1995) [14]. The
using Agar well diffusion method against two organisms viz. results in this study indicate that the inhibitory activity of
Gram-positive organism (Staphylococcus aureus) and both the extracts was comparatively greater than that of
Gram-negative organism (Escherichia coli). The result Gentamicin. The methanolic extracts of P. guajava leaves
indicated that both the methanolic and aqueous extracts showed significant antibacterial activity against S. aureus
prepared from the leaves of Psidium guajava showed and E. coli. Bacterial cells can be killed by the rupture of
inhibitory activity, indicating the susceptibility of both the cell walls and membrane and by the irregular disruption of
micro-organisms towards the extracts. The methanolic the intracellular matrix when treated with plants extracts
extract with a concentration range of (50 mg/ml) and (Kim and Fung., 2004) [9]. The mesh-like peptidoglycan
distilled water extract with a concentration range of (30 layer of the Gram-positive bacteria is more accessible to the
mg/ml) showed a zone of inhibition of 16 mm in diameter, extracts by permeation. Whereas the resistance of the Gram-
while a zone of inhibition of 20 mm was observed with 50 negative bacteria could be attributed to its cell wall structure
mg/ml of Gentamicin concentration (Antibiotic used as a as they are mostly resistant to plant-origin extracts. An
control) against S. aureus as shown in Fig. 4, Table. 3. effective permeability barrier of the Gram-negative bacteria
Whereas the methanolic extract with a concentration range is comprised of a thin lipopolysaccharide membrane, which
of (30 mg/ml) and distilled water extract with a restricts any penetration. But the P. guajava leaf extracts
concentration range of (20 mg/ml) showed a zone of contain active components which show inhibitory effect
inhibition of 17 mm, while a zone of inhibition of 21 mm against Gram-negative bacterium by penetrating its cell wall
was observed with 50 mg/ml of Gentamicin concentration (Biswas
against E. coli as shown in Fig. 5, Table. 4. The size of the
et al., 2013) [3].

Fig 4: Antimicrobial activity of methanolic, distilled water extract, gentamicin against S. aureus

Fig 5: Antimicrobial activity of methanolic, distilled water extract, gentamicin against E. coli

Table 3: Inhibitory action of methanolic, d/w extract and gentamicin on S. aureus

Test organism Zone of inhibition(mm)
Methanolic Extract (50 mg/ml) Distilled water Extract (30 mg/ml) Gentamicin (50 mg/ml)
S. aureus
16 16 20
~ 109 ~
International Journal of Applied Research http://www.allresearchjournal.com

Table 4: Inhibitory action of methanolic, d/w extract and gentamicin on E. coli

Test organism Zone of inhibition(mm)
Methanolic Extract (30 mg/ml) Distilled water Extract (20 mg/ml) Gentamicin (50 mg/ml)
S. aureus
17 17 21

4. Conclusion of phytochemicals. International Journal of Health

This study was undertaken with an aim to determine the Sciences & Research, 2019, 9(2).
phytochemical constituents and the antimicrobial activity of 6. Gonçalves FA, Andrade NM, Bezerra JNS, Macrae A,
guava leaves. The phytochemical analysis showed that P. De Sousa OV, Fonteles-Filho AA et al. Antibacterial
guajava leaves were rich in a wide range of bioactive activity of guava, Psidium guajava Linnaeus, leaf
compounds like phenols, flavonoids, terpenoids, glycosides extracts on diarrhea-causing enteric bacteria isolated
and saponins. Research indicates these bioactive compounds from seabob shrimp, Xiphopenaeus kroyeri (Heller).
to be the active antimicrobial agents in guava leaves. From Revista do Instituto de Medicina Tropical de Sao Paulo.
the results of MIC, it can be suggested that the methanolic 2008; 50(1):11-15.
and aqueous extract produced inhibitory activity with 7. Harley JP, Prescott LM. Laboratory exercises in
varying degrees against both the Gram-positive and Gram- microbiology. Fifth edition. The McGraw –Hill
negative strains. The MIC of the aqueous extract was lower publishing companies, 2002.
than the MIC of the methanolic extract for both S. aureus 8. Khan ZA, Siddiqui MF, Park S. Current and emerging
and E. coli. This indicates that the aqueous extract was more methods of antibiotic susceptibility testing. Diagnostics,
effective than the methanolic extract at inhibiting the growth 2019. (doi:10.3390/diagnostics9020049).
of the micro-organisms. The antimicrobial activity was 9. Kim S, Fung DYC. Antibacterial effect of crude water-
checked, and it was seen that the growth of S. aureus and E. soluble arrow root (Puerariae radix) tea extracts on
coli was inhibited by the methanolic and aqueous extracts food-borne pathogens in liquid medium. Letters in
with visible zones of inhibition using the well diffusion/agar Applied Microbiology. 2004; 39(4):319-325.
cup method. Gentamicin which is a common antibiotic used 10. Lutterodt GD. Inhibition of microlax-induced
for treating various bacterial infections was taken as a experimental diarrhoea with narcotic like extracts of
control and it showed lesser inhibition against the test Psidium guajava leaf in rats. Journal of
organisms as compared to the extracts. Various chronic Ethnopharmacology. 1992; 37(2):151-157.
degenerative diseases have reached epidemic proportions in 11. Maurya A, Maurya SP, Asthana A, Maurya SM,
many countries, increasing the socio-economic burden for Maurya P, Kundu P. Guava (Psidium guajava L.) -
the public health system (Barbalho et al., 2012) [2]. More review article. Indian Journal of Drugs. 2018; 6(4):218-
efforts are being taken to find alternate antimicrobial agents 222.
due to the rapid development of resistance to existing 12. Nayak N, Varghese J, Shetty S, Bhat V, Durgekar T,
agents. Several studies have suggested that products isolated Lobo R et al. Evaluation of a mouthrinse containing
from plant species are preferable compared to synthetic guava leaf extract as part of comprehensive oral care
products. Given this, in the search for a natural regimen- a randomized placebo controlled clinical trial.
antimicrobial agent to fight against human pathogens, P. BMC Complementary and Alternative Medicine, 2019.
guajava leaves could prove to be a good candidate. (https://doi.org/10.1186/s12906-019-2745-8).
However, further studies need to be carried out to determine 13. Ncube NS, Afolayan AJ, Okoh AI. Assessment
the mechanism of P. guajava on other bacterial isolates. techniques of antimicrobial properties of natural
compounds of plant origin: current methods and future
5. References trends. African Journal of Biotechnology. 2008;
1. Abdelrahim SI, Almagboul AZ, Omer MEA, Elegami 7(12):1797-1806.
A. Antimicrobial activity of Psidium guajava L. 14. Power EGM. Aldehydes as biocides. Progress in
Fitoterapia. 2002; 73(7-8):713-715. Medical Chemistry. 1995; 34:149-201.
2. Barbalho SM, Farinazzi-Machado FMV, Goulart RA, 15. Puntawong S, Okonogi S, Pringproa K. In vitro
Brunnati ACS, Ottoboni AMMB, Nicolau CCT. antibacterial activity of Psidium guajava Linn. Leaf
Psidium guajava (guava): a plant of multipurpose extracts against pathogenic bacteria in pigs. CMU. J
medicinal applications. Medicinal and Aromatic Plants, Nat. Sci. 2012, 11(2).
2012, 1(4).
3. Biswas B, Rogers K, Mclaughlin F, Daniels D, Yadav
A. Antimicrobial activities of leaf extracts of guava
(Psidium guajava L.) on two gram-negative and gram-
positive bacteria. International Journal of Microbiology,
2013. (http://dx.doi.org/10.1155/2013/746165).
4. Chukiatsiri K, Tippaya K, Ngasaman R. Effectiveness
of pomegranate rind (Punica granatum Linn.) and guava
leaf extract (Psidium guajava Linn.) for inhibition of
multidrug resistant Escherichia coli recovered from
diarrhoeal piglets. Journal of Science and Technology,
2019. SJST-2019-0189.R1.
5. Das M, Goswami S. Antifungal and antibacterial
property of guava (Psidium guajava) leaf extract: role

~ 110 ~